KR20160027095A - Method for manufacturing a shaped cross-linked hyaluronic acid product - Google Patents

Abstract

The process for making a shaped crosslinked hyaluronic acid product comprises reacting a non-crosslinked precipitated hyaluronic acid substrate of the desired shape with an amount of at least one polyfunctional crosslinking agent (s) having a pH of 11.5 or higher and providing precipitation conditions for hyaluronic acid In a liquid medium comprising at least one organic solvent (s), wherein the degree of modification is from 1 to 40 units of crosslinking agent per 1000 units of disaccharide, under conditions suitable to obtain a precipitated, shaped crosslinked hyaluronic acid product .

Description

METHOD FOR MANUFACTURING A SHAPED CROSS-LINKED HYALURONIC ACID PRODUCT [0002]

The present invention relates to the field of polysaccharides. More particularly, the present invention relates to a novel method of crosslinking hyaluronic acid and a novel method of making a shaped crosslinked hyaluronic acid product.

One of the most widely used biocompatible polymers for medical use is hyaluronic acid. It is a naturally occurring polysaccharide belonging to the glycosaminoglycan (GAG) group. Hyaluronic acid and other GAGs are negatively charged heterosaccharide chains with the ability to absorb large amounts of water. The products of hyaluronic acid and hyaluronic acid are widely used in biomedical and cosmetic fields, for example viscosurgery and as skin fillers.

Absorbent gels or hydrogels are widely used in the biomedical field. These are generally prepared by chemically crosslinking the polymer to an infinite network. The natural hyaluronic acid and partially crosslinked hyaluronic acid products absorb water until they are completely dissolved, but the crosslinked hyaluronic acid gels typically remain in water until saturated, i.e., until they have a defined liquid retention capacity or swelling degree , Absorbing a certain amount of water.

In preparing gels from biocompatible polymers, it is advantageous to ensure a low degree of crosslinking to maintain high biocompatibility. However, often thicker gels are needed to have the appropriate biomedical effect, and in such cases, biocompatibility will often be lost.

Since hyaluronic acid is present in most living organisms with the same chemical structure except molecular weight, this provides minimal response and allows for advanced medical uses. Crosslinking and / or other modifications thereof are needed to improve the degradation resistance or residence time of hyaluronic acid molecules in vivo. Moreover, this modification affects the liquid retention capacity of the hyaluronic acid molecule. As a result, hyaluronic acid has been the object of many modification attempts.

Because the crosslinked hyaluronic acid gel products are highly complex chemical structures, they are typically characterized by a combination of their chemical structure and their physical properties. The deflection of chemical structures from unmodified hyaluronic acid is typically reported as strain, strain, cross-linking, cross-linking index or chemical strain, all of which relate to the amount of cross-linking agent covalently bonded to hyaluronic acid. Throughout this disclosure, the term variants will be used. The most relevant physical properties of the crosslinked hyaluronic acid gel product are the volume of liquid that the gel can absorb and the rheological properties of the gel. Both properties describe the structural stability of the gel, which is often referred to as gel strength or rigidity, but the absorption of the liquid can be ascertained for the dry gel while the rheological properties should be measured on the gel swelling to the desired concentration . Typical expressions for liquid absorption are swelling, swelling capacity, liquid retention capacity, swelling degree, degree of swelling, maximum liquid uptake rate and maximum swelling degree. Throughout this disclosure, the term swelling degree will be used. With respect to the rheological properties of the crosslinked hyaluronic acid gel product, oscillatory rheometry is essential to ascertain the fluidity of the gel, whereas rotational rheometry alone is useful for ascertaining the fluidity of the liquid. These measurements provide the resistance of the gel to deformation in units of modulus and viscosity. The higher gel strength will provide greater resistance to deformation of the swollen gel product to the desired concentration.

US 2007/0066816 discloses a process for the preparation of double-crosslinked hyaluronic acid comprising the step of crosslinking the hyaluronic acid substrate in step 2 using epoxides and carbodiimides, respectively.

EP 2 199 308 A1 discloses the crosslinking of hyaluronan powder dispersed in a liquid medium containing ethanol. The resulting product has a poorly controlled shape.

US 2012/0034462 A1 suggests that cross-linked HA gels can be made into thin strands by passing solid material of cross-linked HA gel through a sieve or mesh, without experimental evidence.

Despite advances in the art, there is a need for another method of making a shaped crosslinked hyaluronic acid product that maintains biocompatibility but has an appropriate liquid retention capacity and degradation profile. In particular, it is desirable to minimize the strain required to obtain a molded hyaluronic acid gel product with a desired gel strength that can be measured, for example, as a liquid retention capacity.

Some known soft tissue augmentation treatments, such as implants, often suffer from the disadvantage that the implant or a portion thereof moves away from the desired treatment site. Another problem with some known tissue augmentation procedures, such as implants, is that the implant is displaced from the desired treatment site. Implant movements and displacements are disadvantageous to the patient because they can affect the cosmetic and / or therapeutic outcome of the procedure and interfere with removal of the implant when required. It is very beneficial to maintain the integrity and position of the implant for a desired period of time. To avoid these problems, the gel needs to have a specific gel strength to resist deformation. This characteristic can be measured by using the flow measurement in a vibrational manner.

It is an object of the present invention to provide a process for producing a crosslinked hyaluronic acid product having a desired shape. A particular aim is to provide a process for producing a crosslinked hyaluronic acid product having a shape that limits the likelihood of product migration after implantation on an individual.

It is a further object of the present invention to provide a process for the production of shaped crosslinked hyaluronic acid products which have high biocompatibility, i. E. Maintain high biocompatibility of hyaluronic acid when fixed in a preferred and useful shape.

In order to achieve these basic objects and / or other objects to be ascertained from the present specification, a basic object of the present invention is to provide a liquid container having a high gel strength as confirmed by a low liquid holding capacity to a medium liquid holding capacity or swelling degree, It has been realized that a method for producing a molded crosslinked hyaluronic acid product having a low to intermediate degree of deformation is provided.

A further object of the present invention is to provide a process for the production of shaped crosslinked hyaluronic acid products, wherein the liquid retention capacity can be controlled or influenced by other parameters than the degree of hyaluronic acid deformation.

It is an object of the present invention to provide a method of making a shaped crosslinked hyaluronic acid product wherein a large part of the combined crosslinking agent (s) is (at least) connected at two ends, i. E., Achieving high crosslinking efficiency.

A further object of the present invention is to provide a shaped crosslinked hyaluronic acid product having a low to moderate degree of deformation and at the same time having a low liquid retention capacity, a degree of swelling or moderate liquid retention capacity or swelling.

It is an object of the present invention to provide a shaped crosslinked hyaluronic acid product with high deformation resistance.

In these and other objects to be ascertained from this disclosure, the present invention provides, according to a first aspect, a process for the production of a shaped crosslinked hyaluronic acid product,

(i) providing a hyaluronic acid substrate dissolved in a first liquid medium which is an aqueous solution, without crosslinking;

(ii) precipitating a hyaluronic acid matrix by treating the hyaluronic acid matrix with a second liquid medium, wherein the second liquid medium comprises, without crosslinking, at least one first water-soluble organic solvent (s) in an amount to provide precipitation conditions for hyaluronic acid ; And

(iii) contacting the non-crosslinked precipitated hyaluronic acid substrate of the desired shape with one or more second organic solvent (s) having a pH of at least 11.5 and at least one multifunctional crosslinking agent (s) and an amount to provide precipitation conditions for hyaluronic acid Crosslinked hyaluronic acid product, in a third liquid medium containing a crosslinked hyaluronic acid,

Step (i) and / or step (ii) further comprise arranging the hyaluronic acid substrate in the desired shape.

The method has been found to advantageously enable the production of molded crosslinked hyaluronic acid products with highly desirable properties. The present process provides good control of crosslinking, since crosslinking occurs only in the solid (precipitated) state and does not occur between dissolved states and / or process steps. The resulting product is unique in that it is a gel with a low degree of swelling despite low deformation of hyaluronic acid. It is surprising that gel products with limited swelling can be obtained at these low strains. It is also surprising that processes with a single cross-linking reaction can achieve products with these desired properties. Among many applications, this method enables the production of crosslinked hyaluronic acid products with a predetermined shape to be maintained during the manufacturing process. The present method also enables the production of biocompatible shaped crosslinked hyaluronic acid products.

In certain embodiments, the first two steps (i) and (ii) are conducted in the absence of a cross-linking agent, and the multi-functional cross-linking agent is added to the third cross-linking step (iii). This ensures that the amount of cross-linking agent is highly regulated since the cross-linking agent reacts or is lost during previous steps, for example during the precipitation step.

In one embodiment, step (i) further comprises the step of arranging the hyaluronic acid substrate solution in a desired shape on the hydrophobic surface; Precipitation of the hyaluronic acid matrix formed in step (ii) occurs on the hydrophobic surface. This is advantageous in preventing the buildup of the structures and maintaining their shape. The hydrophobic surface is preferably selected from fluorocarbons, polypropylene (PP), polyethylene terephthalate glycol-modified (PETG), polyethylene (PE) and polytetrafluoroethylene (PTFE).

In some embodiments, the aqueous solution of step (i) comprises 40-100% by volume of water and 0-60% by volume of lower alkyl alcohol (s). Thereby, an entangled structure can be achieved, which will probably be advantageous for obtaining a gel product with the desired properties.

In a specific embodiment, the second liquid medium of step (ii) comprises 0 - 30% by volume of water and 70 - 100% by volume of the first water soluble organic solvent (s). In some embodiments, the second liquid medium of step (ii) comprises 0-10 volume% water of water and 90-100 volume% of the first water soluble organic solvent (s). The high concentration of the first water-soluble organic solvent (s) is considered to be advantageous for achieving rapid precipitation. Thereby, a twisted structure can be achieved, which may be advantageous to obtain a gel product with the desired properties.

In certain embodiments, the first water soluble organic solvent (s) of step (ii) is one or more lower alkyl alcohol (s). In some embodiments, the lower alkyl alcohol is ethanol. These organic solvents provide rapid precipitation.

Step (i) and / or step (ii) further comprise arranging the hyaluronic acid substrate in the desired shape. The terms "molded" and "desired shape" refer to intended designs that are useful in the final product, i.e. not simply lyophilized or precipitated hyaluronan powder. In certain embodiments, the shape is selected from the group consisting of particles, fibers, strings, strands (preferably, at least one dimension) having an extension at least 0.5 mm, preferably greater than 1 mm, more preferably greater than 5 mm strand, net, film, disk, and bead. In some embodiments, the shape of the substrate has an elongation at at least one dimension of less than 5 mm, preferably less than 1 mm. This facilitates the accessibility of the crosslinker (s) to many of the available binding sites of the precipitated hyaluronic acid product in a subsequent crosslinking step. In certain embodiments, the shape of the substrate is elongated in the longitudinal direction, wherein the ratio of the longitudinal elongation of the elongate body to the elongated transverse elongation is at least 5: 1, such as at least 10: 1, such as at least 20: 1, for example less than or equal to 25 000: 1, such as less than or equal to 100: 1. Since the longitudinally stretched shape is retained in the resulting product during the entire method, the crosslinked product can be designed to be easily injectable, while preventing or reducing movement / displacement in vivo. In a specific embodiment, the shape is a fiber and the ratio between its length and its average diameter is at least 5: 1, such as at least 10: 1, such as at least 20: 1, alternatively at most 100: 000: 1 or less, for example, 100: 1 or less.

In certain embodiments, step (ii) comprises extruding the hyaluronic acid matrix into a second liquid medium comprising the first water soluble organic solvent (s) in an amount to provide precipitation conditions for hyaluronic acid, And allowing the substrate to form precipitated fibers in the second liquid medium.

In some embodiments, the third liquid medium of step (iii) comprises 0-35 vol% water, 65-100 vol% of the second organic solvent (s) and one or more multifunctional crosslinker (s). In certain embodiments, the second organic solvent (s) of step (iii) is one or more lower alkyl alcohol (s). In some embodiments, the lower alkyl alcohol is ethanol.

In a specific embodiment, the third liquid medium of step (iii) has a pH of 13 or higher. It has surprisingly been realized that carrying out cross-linking on a shaped substrate precipitated at an elevated pH provides a molded gel product with efficient cross-linking, i.e., a low degree of deformation provides a hard gel with a low degree of swelling.

In a specific embodiment, the multifunctional crosslinking agent (s) are individually selected from the group consisting of divinyl sulfone, multiepoxide and diepoxide. In some embodiments, the multifunctional crosslinker (s) is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxy octane Respectively. In certain embodiments, the multifunctional crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

In certain embodiments, the at least one multifunctional crosslinker (s) provides a single type of crosslinking. In a specific embodiment, the at least one multifunctional crosslinker (s) provides stable ether crosslinking. The present method advantageously provides a single crosslinked hyaluronic acid gel product that is stable and readily sterile, e.g., autoclaved.

In some embodiments, the method further comprises:

(iv) treating the precipitated, crosslinked hyaluronic acid product in non-precipitation conditions; And

(v) separating the crosslinked hyaluronic acid product into a non-precipitated form.

In certain embodiments, step (v) further comprises sterilizing the crosslinked hyaluronic acid product.

According to another aspect, the present invention provides a shaped crosslinked hyaluronic acid product wherein the degree of modification is from 1 to 40 units of crosslinking agent per 1000 units of disaccharide and the degree of swelling is from 4 mL to 300 mL per g of hyaluronic acid. Such shaped cross-linked hyaluronic acid products have very useful properties, including low to moderate strain, and at the same time a unique combination of low to medium swelling or liquid retention capacity. Thus, it is possible to provide a hard, crosslinked hyaluronic acid product having a desired shape while maintaining the inherent biocompatibility of the hyaluronic acid. The crosslinked hyaluronic acid product according to the present invention is designed to have a predetermined shape or structure.

In certain embodiments, the degree of swelling is 15-180 mL of hyaluronic acid per gram of sugar.

The strain efficiency (MoE) is a measure of the ratio between the minimum HA concentration (C _min ) or stiffness / strength of the gel and the chemical strain by the cross-linking agent (s). In a specific embodiment, the strain efficiency is at least 10. In some embodiments, the strain efficiency is in the range of 20-190 or 20-150. Products with a strain efficiency of greater than or equal to 10, such as 20-190 or 20-150, are initially combined with low to moderate strains, while at the same time combined with low to moderate swelling or liquid retention capacity. It is thereby possible to provide a hard, crosslinked hyaluronic acid product of the desired shape, which is biocompatible and has high deformation resistance.

In certain embodiments, the ratio of cross-linkers that describe the ratio of the total bonded cross-linker linking two (or more) disaccharides is greater than 35%. In a specific embodiment, the ratio of crosslinker is at least 40%, and in some cases even at least 50%. These molded products consequently have a low number of crosslinks which do not provide effective crosslinking in the products. High crosslinking ratios enable surprisingly low total deformation with respect to low to moderate swelling, which is advantageous in terms of biocompatibility but is a combination sufficient to maintain the desired shape.

In some embodiments, the shaped hyaluronic acid product is crosslinked with one or more multifunctional crosslinking agent (s) that are individually selected from the group consisting of divinyl sulfone, multiepoxide, and diepoxide. In certain embodiments, the multifunctional crosslinker (s) is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE), and diepoxy octane Respectively. In a specific embodiment, the multifunctional crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

In certain embodiments, the shaped hyaluronic acid product is single crosslinked. Single-bridged products have chemically well-defined advantages. It is advantageous for the shaped product with a single type of cross-link to exhibit this desirable property. In some embodiments, the shaped hyaluronic acid product is multiple crosslinked. In a specific embodiment, the shaped hyaluronic acid product is crosslinked by ether crosslinking. The shaped ether crosslinked hyaluronic acid gel product according to the invention is stable and can be easily sterilized, for example, autoclaved.

In certain embodiments, the hyaluronic acid product has a shape selected from the group consisting of particles, fibers, strings, strands, nets, films, discs and beads. In some embodiments, the hyaluronic acid product is hollow and has several layers. In certain embodiments, the crimp is elongated in the longitudinal direction and the ratio of its longitudinal stretch to its longest longitudinal stretch is at least 5: 1, such as at least 10: 1, such as at least 20: 1, 000: 1 or less, for example, 25,000: 1 or less, for example, 100: 1 or less. The crosslinked product stretched in the longitudinal direction can be designed to be easily injectable, although it prevents or reduces movement / displacement in vivo. In a specific embodiment, the hyaluronic acid product is a fiber and the ratio between its length and its width, for example its average diameter, is at least 5: 1, such as at least 10: 1, for example at least 20: : 1 or less, for example 25,000: 1 or less, for example, 100: 1 or less.

In some embodiments, the shaped hyaluronic acid product is in a fully swollen state. In its fully swollen state, the product has a longitudinally elongated shape and its longest transverse elongation is preferably less than 5 mm, for example less than 1.5 mm, preferably less than 0.2 mm. Longitudinally elongated crosslinked products having a longest transverse elongation of less than or equal to 5 mm are readily injectable. Moreover, in its fully swollen state, the product preferably has a longitudinally elongated shape and its longitudinal stretch is preferably greater than 2 mm, for example greater than 25 mm, such as greater than 500 mm. A longitudinally stretched crosslinked product having a longitudinal elongation of 2 mm or more is advantageous because it prevents or reduces movement / displacement in vivo.

In other embodiments, the shaped hyaluronic acid product is in a partially swollen or non-swollen state.

In a specific embodiment, the shaped hyaluronic acid product is autoclavable. In a further specific embodiment, the shaped hyaluronic acid product is autoclaved.

One of the preferred methods of making the shaped crosslinked hyaluronic acid product according to the invention is by the process according to the invention.

According to yet another aspect, the present invention provides an aqueous composition comprising a shaped crosslinked hyaluronic acid product according to the present invention, and optionally a buffer.

In certain embodiments, the shaped hyaluronic acid product according to the invention or the aqueous composition according to the invention is useful as a medicament or a medical device in a medical or surgical way.

According to a further aspect, the invention provides the use of a shaped crosslinked hyaluronic acid product according to the invention or of an aqueous composition according to the invention for cosmetic or medical surgery. That is, the present invention provides a molded crosslinked hyaluronic acid product according to the present invention or an aqueous composition according to the present invention for use in cosmetic surgery or medical surgery.

In some embodiments, the use is a cosmetic surgery selected from skin rejuvenation and human contouring. In some other embodiments, the use is a medicament in a therapeutic and / or medical operation selected from skin rejuvenation, human contouring, prevention of tissue adhesion, channel formation, incontinence treatment, and orthopedic application.

According to one aspect, the present invention provides the use of a shaped crosslinked hyaluronic acid product according to the invention or an aqueous composition according to the invention in drug delivery. In other words, the present invention provides a shaped crosslinked hyaluronic acid product according to the invention or an aqueous composition according to the invention for use in drug delivery.

According to a still further aspect, the present invention provides a pre-filled syringe pre-filled with a sterile, shaped crosslinked hyaluronic acid product according to the invention or a sterile aqueous composition according to the invention.

According to one aspect, the present invention provides a method of treating a subject suffering from a cosmetic or surgical procedure, comprising the step of administering a shaped crosslinked hyaluronic acid product according to the invention or an aqueous composition according to the invention to a subject in need thereof Provide a treatment method.

In certain embodiments, the subject is subjected to a cosmetic surgery selected from skin rejuvenation and human contouring. In certain embodiments, the subject is undergoing medical surgery or medical treatment for a condition selected from skin allergy, human contouring, prevention of tissue adhesion, channel formation, incontinence treatment, and orthopedic application.

Figure 1 shows a fully swollen crosslinked HA net.
Figure 2 shows a 400 MHz < ¹ > H NMR spectrum of two enzymatically degraded HA gel formulations.
Figure 3 shows a photomicrograph of a cross-linked HA gel fiber.
4 is a graph showing the change in swelling degree (SwD) in g / g during storage at 60 DEG C for 14 days.
Figure 5 shows a micrograph of a crosslinked HA powder (comparative example) and a product according to the invention.
Figure 6 shows a micrograph of a crosslinked HA gel fiber.
Figure 7 shows a micrograph of a cross-linked HA gel (comparative example) once passed through a mesh screen.

According to one aspect, the present invention provides a manufacturing method. The method is for producing a crosslinked hyaluronic acid product molded from a hyaluronic acid substrate.

Unless otherwise stated, the term "hyaluronic acid" encompasses a wide variety of chain lengths and charge states as well as combinations of all variants and variants of hyaluronic acid or hyaluronan with various chemical modifications. That is, the term also includes various hyaluronate salts of hyaluronic acid, such as sodium hyaluronate. Various modifications of hyaluronic acid, such as oxidation, for example oxidation of -CH ₂ OH groups to -COOH, Periodate oxidation of vicinal hydroxy groups, selective reduction or imine formation, etc .; For example, reduction to the -COOH -CH ₂ OH; sulfuration; Deamidation, and optionally followed by de-amination or amide formation with a new acid; Esterification; Substitution with various compounds, e. G., By cross-linking agents or carboymides; Coupling of different molecules such as proteins, peptides and active drug components to hyaluronic acid; And deacetylation are also encompassed by this term. Those skilled in the art are well aware that various forms of hyaluronic acids have different chemical properties that must be considered during chemical modification and analysis. For example, if it is desired to obtain a hyaluronic acid solution having a particular pH, both the acidity of the substance to be dissolved, the acidity of the soluble liquid, and any buffering capacity will affect the resulting pH of the solution.

It is preferred that the hyaluronic acid substrate is a hyaluronic acid or hyaluronate salt that has not undergone chemical modification, i. E. Has not undergone crosslinking or other modification prior to the practice of the present preparation process.

Hyaluronic acid can be obtained from a variety of sources of animal and non-animal origin. Sources of non-animal origin include yeast, preferably bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 1.5 MDa to 3 MDa, but other molecular weight ranges, such as 0.5 MDa to 10 MDa, are also possible.

The product produced by the present process is shaped crosslinked hyaluronic acid. The present method provides cross-linking between these chains when the hyaluronic acid chains are arranged in their preferred shape, which cross-links the covalent bonds, the physical twist of the hyaluronic acid chains and various interactions such as hydrogen bonding, Van der Waals attraction and Form a continuous shaped network of hyaluronic acid molecules that are held together by electrostatic interactions. The shaped crosslinked hyaluronic acid product according to the present invention is a gel or a hydrogel. That is, the hyaluronic acid product may be considered to be water-insoluble, but when placed in a liquid, typically an aqueous liquid, it can be considered to be a substantially diluted crosslinking system of hyaluronic acid molecules.

The resulting shaped crosslinked hyaluronic acid product is preferably biocompatible. This means that no or only very slight immune response occurs in the treated individual. In an embodiment, there is provided a method for confirming the biocompatibility of the hyaluronic acid product, and a result of testing the biocompatibility of the crosslinked hyaluronic acid product according to the present invention in a rat.

The process according to the invention comprises three or more steps: a preparation step, a precipitation step and a crosslinking step. In certain embodiments, the method comprises these three steps.

In a first method step, a hyaluronic acid substrate is provided. As noted above, the term "hyaluronic acid substrate" includes any combination of mutants and variants of hyaluronic acid or hyaluronan with various chain lengths and charge states as well as various chemical modifications. The hyaluronic acid substrate is a chemically unmodified hyaluronic acid or hyaluronate salt having an average molecular weight in the range of 0.5-10 MDa, preferably 0.8-5 MDa, more preferably 1.5-3 MDa or 2-3 MDa. , And preferably sodium hyaluronate. It is preferred that hyaluronic acid is obtained from a non-animal origin, preferably from bacteria.

The hyaluronic acid substrate is dissolved in a first liquid medium which is an aqueous solution. The terms "dissolved" and "solution" are understood to mean that the hyaluronic acid substrate is present in a homogeneous mixture with the liquid and that an energetically favorable interaction occurs in the mixture. Addition of the liquid to the solution lowers the concentration of the hyaluronic acid substrate to be dissolved. The solution is aqueous, i.e. the solution comprises water. The aqueous solution may simply consist of a hyaluronic acid substrate dissolved in water. The aqueous solution preferably comprises 40-100% by volume of water and 0-60% by volume of lower alkyl alcohol (s). The term "lower alkyl alcohol" includes primary, secondary and tertiary alkyl alcohols having from 1 to 6 carbon atoms, i.e., C _1-6 alkyl alcohols. Specific examples of lower alkyl alcohols include methanol, ethanol, denatured spirit, n-propanol, isopropanol, n-butanol, isobutanol, and t-butanol. Preferred lower alkyl alcohols are methanol and ethanol, especially ethanol, due to their price, availability and ease of handling. The concentration of the lower alkyl alcohol preferably ranges from 0-40%, such as 0-20%, 10-30%, or 20-40% ethanol and is adjusted accordingly using a water component. The pH of the aqueous solution is suitably 6 or more, for example, 9 or more.

Alternatively, the first method step comprises arranging the hyaluronic acid substrate into a desired shape, such as particles, fibers, strings, strands, nets, films, discs and beads, optionally comprising layers of hollow or different materials . This can be achieved in a variety of ways, for example by molding or extrusion. Extrusion of the hyaluronic acid substrate typically involves passing the hyaluronic acid substrate solution through a desired sized opening. The extruded hyaluronic acid substrate spontaneously forms precipitated fibers, strings or strands. The dimensions, e.g., thickness, of the fibers, strings or strands may be varied, for example using various opening diameters in the range of 0.1-2 mm or 14-30 G, extrusion pressure, extrusion rate and / or hyaluronic acid concentration, Dimensions, or types. By using different types of orifices and chinks, different shapes or structures can be created. For example, hyaluronic acid can be deposited as a film, net, disk or bead.

In a preferred embodiment, the hyaluronic acid substrate solution is arranged on the hydrophobic surface in the desired shape; Subsequently, precipitation of the molded hyaluronic acid substrate occurs on the hydrophobic surface. This is advantageous for preventing the build-up of the shape structure and maintaining the desired shape until the shape is fixed by a subsequent cross-linking step. Suitable hydrophobic surfaces are well known to those skilled in the art and include, for example, fluorocarbons, polypropylene (PP), polyethylene terephthalate glycol-modified (PETG), polyethylene (PE) and polytetrafluoroethylene do.

This shape can be maintained throughout the manufacturing process and in the final product. When the hyaluronic acid substrate is present in precipitated form, it is preferred that the shape has an elongation of at least one dimension less than 5 mm, preferably less than 1 mm, such as less than 0.5 mm, or even less than 0.2 mm. This facilitates the accessibility of the crosslinker (s) to many of the available binding sites of the precipitated hyaluronic acid product in a subsequent crosslinking step. Also, the shape is elongated in the longitudinal direction and its longitudinal elongation: the ratio of its longest longitudinal elongation is at least 5: 1, such as at least 10: 1, for example at least 20: For example, 25 000: 1 or less, for example, 100: 1 or less. Since the longitudinally stretched shape is maintained throughout the process and in the resulting product, the crosslinked product can be designed to be easily injectable, while preventing or reducing movement / displacement in vivo. A suitable example of such a shape is a fiber and the ratio between its length and its width is at least 5: 1, such as at least 10: 1, such as at least 20: 1 and optionally at most 100: 1 or less, for example, 100: 1 or less. A preferred composition comprises a crosslinked strand / fiber-shaped hyaluronic acid product according to the invention wherein more than 50% of the product has a ratio of its longitudinal elongation to its longest longitudinal elongation of at least 5: 1, For example, not less than 10: 1, for example not less than 20: 1, alternatively not more than 100,000: 1, for example not more than 25,000: 1, for example not more than 100:

For example, a crosslinked hyaluronic acid product according to the present invention having a single-stranded or fibrous shape filling a syringe of 20 mL or less has a thickness of 1 mm and a length of 25 m in its swollen state, i.e., its longitudinal elongation: The ratio of the longest transverse elongation thereof may be 25000: 1. An example of a preferred composition comprises a crosslinked strand / fiber-shaped hyaluronic acid product according to the invention wherein more than 50% of the product has a longitudinal elongation of greater than 2 mm and a longest transverse elongation of less than 0.2 mm That is, the ratio is 10: 1 or more.

The first method step is carried out without crosslinking, which can be accomplished by omitting the crosslinking agent at this stage and / or providing conditions unsuitable for crosslinking. It is important to prevent cross-linking from occurring until a desired shape is obtained. This is advantageous in obtaining and maintaining the final product of the desired shape, since the shaping of the substrate is not limited by conventional crosslinking, and all the crosslinks produced in the third step are about maintaining the desired shape of the product to be. The first step is preferably carried out in the absence of a crosslinking agent. This provides good control that does not occur between cross-linked dissolved states and / or process steps. Also, this ensures that the amount of cross-linking agent is tightly controlled and that the resulting product is homogeneous in quality, since the cross-linking agent does not react or lose in previous steps. The prevention of crosslinking, especially the addition of crosslinking agents at this stage, is useful for obtaining an appropriate manufacturing process to scale up to an industrial scale and provide products with homogeneous quality.

In the second method step, the hyaluronic acid substrate precipitates due to the reduced solubility of the hyaluronic acid substrate. This is accomplished by treating the hyaluronic acid substrate with a second liquid medium that is insoluble in the substrate. The second liquid medium comprises the one or more first water soluble organic solvent (s) in an amount to provide precipitation conditions of hyaluronic acid. The resulting solid precipitate comes off the solute phase and can be separated from the residual liquid, typically by filtration, decanting, centrifugation, or passively using a pair of tweezers or the like. In one preferred embodiment, the precipitated hyaluronic acid substrate is also removed from the medium and dried. The precipitate can also be maintained in suspension in the second liquid medium. Thus, in another preferred embodiment, the precipitated hyaluronic acid substrate is not subjected to drying. It is advantageous to precipitate the hyaluronic acid substrate in a rapid manner, for example by extruding or immersing the substrate in a second liquid medium in which the hyaluronic acid substrate is insoluble.

The organic solvent used according to the present invention is a carbon-containing solvent and can exhibit various polarities. Although referred to as "solvent ", it should be understood that these organic solvents are used to balance and transfer the solubility of hyaluronic acid during the manufacturing process. Hyaluronic acid can dissolve very well in the organic solvent at certain organic solvent concentration intervals, but when the organic solvent concentration increases, it forms a precipitate. For example, hyaluronic acid may dissolve in an organic solvent, such as a lower alkyl alcohol and a 50/50 (vol / vol) mixture of water, but it will separate and form a precipitate in a 90/10 (vol / vol) mixture . When subjected to a non-precipitation condition, for example, when treated in a 50/50 or 0/100 mixture, the hyaluronic acid returns to the non-precipitated, dissolved state. Those skilled in the art are well aware that other factors such as temperature, pH, ionic strength, and the type of organic solvent (s) may affect the concentration of organic solvent (s) that limit the precipitation of hyaluronic acid. The limiting concentration for precipitation of hyaluronic acid under given conditions is well known or can be readily ascertained by one of ordinary skill in the art. For example, the limiting concentration for precipitation of hyaluronic acid (in a mixture of water and ethanol) is about 70% ethanol.

But are not limited to, organic solvents according to the present invention include but are not limited to pentane, hexane, cyclohexane, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, ethyl acetate, Diethyl ether, tetrahydrofuran, acetonitrile, methyl ethyl ketone, acetone, lower alkyl alcohols such as methanol, ethanol, propanol, isopropanol and butanol. The organic solvent according to the present invention is preferably water-soluble. A preferred group of organic solvents is lower alkyl alcohols. The term lower alkyl alcohol includes primary, secondary and tertiary alkyl alcohols having from 1 to 6 carbon atoms, i.e., C _1-6 alkyl alcohols. Specific examples of the lower alkyl alcohol include methanol, ethanol, modified distillation column, n-propanol, isopropanol, n-butanol, isobutanol and t-butanol. Preferred lower alkyl alcohols are methanol and ethanol, especially ethanol, due to their price, availability and ease of handling.

The second liquid medium of the second process step is suitably comprised as an aqueous medium, i.e. to some extent, water. The second liquid medium comprises from 0 to 30% by volume of water and from 70 to 100% by volume of the first water soluble organic solvent (s), preferably from 0 to 10% by volume of water and from 90 to 100% by volume of the first water soluble organic solvent (s) . In certain embodiments, the concentration of the first water soluble organic solvent (s) may be as high as 95%, such as 99% or even 99.5%, such as 99%, of methanol or ethanol. The high concentration of the first water-soluble organic solvent (s) is considered to be advantageous for achieving rapid precipitation. Thereby, a twisted structure can be achieved, which will probably be advantageous for obtaining a gel product with desirable properties.

The second method step is carried out without crosslinking, which can be achieved by omitting the crosslinking agent at this stage and / or by providing an inappropriate condition for crosslinking. It is important to prevent cross-linking from occurring until a desired shape is obtained. This is advantageous in obtaining and maintaining the final product of the desired shape, since the shaping of the substrate is not limited by conventional crosslinking, and all the crosslinks produced in the third step are about maintaining the desired shape of the product to be. The second step is preferably carried out in the absence of a crosslinking agent. This provides good control that does not occur between cross-linked dissolved states and / or process steps. Also, this ensures that the amount of cross-linking agent is tightly controlled and that the resulting product is homogeneous in quality, since the cross-linking agent does not react or lose in previous steps. The prevention of crosslinking, especially the addition of crosslinking agents at this stage, is useful for obtaining an appropriate manufacturing process to scale up to an industrial scale and provide products with homogeneous quality.

Alternatively, the second method step comprises arranging the hyaluronic acid substrate into a desired shape such as particles, fibers, strings, strands, nets, films, discs and beads, optionally comprising layers of hollow or different materials . This can be achieved in a variety of ways, for example by molding or extrusion. This shape can be maintained throughout the manufacturing process and in the final product. When the hyaluronic acid substrate is present in precipitated form, the shape of the precipitated substrate preferably has an elongation at least in one dimension of less than 5 mm, preferably less than 1 mm, such as less than 0.5 mm or even less than 0.2 mm . This facilitates the accessibility of the crosslinker (s) to many of the available binding sites of the precipitated hyaluronic acid product in a subsequent crosslinking step. Further, the shape of the precipitated substrate is elongated in the longitudinal direction, and the ratio of the elongation in the longitudinal direction thereof to the elongated transverse elongation in the longitudinal direction is not less than 5: 1, for example, not less than 10: 1, for example, not less than 20: It is preferably 100 000: 1 or less, for example, 25 000: 1 or less, for example, 100: 1 or less. Since the longitudinally stretched shape is maintained throughout the process and in the resulting product, the crosslinked product can be designed to be easily injectable, while preventing or reducing movement / displacement in vivo. A suitable example of such a shape is a fiber and the ratio between its length and its width is at least 5: 1, such as at least 10: 1, such as at least 20: 1 and optionally at most 100: 1 or less, for example, 100: 1 or less. For example, a crosslinked hyaluronic acid product according to the present invention having a single-stranded or fibrous shape filling a syringe of 20 mL or less has a thickness of 1 mm and a length of 25 m in its swollen state, i.e., its longitudinal elongation: The ratio of the longest transverse elongation thereof may be 25000: 1.

The second method step may comprise extruding the hyaluronic acid substrate into a second liquid medium comprising the first water soluble organic solvent (s) in an amount to provide precipitation conditions. This typically involves passing the hyaluronic acid substrate solution through a desired sized opening into the second liquid medium. The extruded hyaluronic acid substrate spontaneously forms precipitated fibers, strings or strands in the second liquid medium. The dimensions, e.g., thickness, of the fibers, strings or strands may be varied, for example using various opening diameters in the range of 0.1-2 mm or 14-30 G, extrusion pressure, extrusion rate and / or hyaluronic acid concentration, Dimensions, or types. By using different types of holes and gaps, different shapes or structures can be created. For example, hyaluronic acid can be deposited as a film, net, disk or bead. When fibers, strings or strands are formed, the ratio between their length and their average diameter is at least 5: 1, such as at least 10: 1, such as at least 20: 1, alternatively at most 100: 000: 1 or less, for example, 100: 1 or less. Advantages of the fiber / string / strand configuration are that the fibers / strings / strands themselves can be twisted at macroscopic levels, such twisting leading to a coil or ball effect, which for example maintains the integrity of the implant It is advantageous to do.

In the third method step, the precipitated hyaluronic acid substrate initially undergoes crosslinking in the third liquid medium. Refers to introducing a stable covalent bond (bridging) between (at least) two distinct hyaluronic acid chains or (at least) two distinct sites of a single hyaluronic acid chain, Thereby forming a continuous network. The crosslinking may simply be a covalent bond between two atoms of the hyaluronic acid chain, for example, an ether bond between two hydroxyl groups, or an ester bond between one hydroxy group and one carboxyl group. Crosslinking may also be a linker molecule covalently bonded to two or more atoms of different hyaluronic acid chains, or individual moieties of a single hyaluronic acid chain. While crosslinking can occur spontaneously under certain conditions, crosslinking typically involves the use of one or more crosslinking agents to facilitate and accelerate this process. If cross-linking is achieved, the cross-linking agent (s) can be completely or partially linked to the hyaluronic acid, or the cross-linking agent (s) can be degraded. Any remaining residues of the non-bonded cross-linker can be removed after crosslinking.

It is important to prevent cross-linking from occurring until a desired shape is obtained. This is advantageous in obtaining and maintaining the final product of the desired shape, since the shaping of the substrate is not limited by conventional crosslinking, and all the crosslinks produced in the third step are about maintaining the desired shape of the product to be. The first two steps are preferably carried out in the absence of a cross-linking agent, and the cross-linking agent is preferably added in the third cross-linking step. This provides good control where crosslinking occurs only in the solid (precipitated) state and does not occur between dissolved states and / or process steps. This also ensures that the amount of cross-linking agent is strictly controlled and the resulting product is homogeneous in quality, since the cross-linking agent does not react or lose during previous steps, for example during the precipitation step. In addition, focusing on crosslinking, especially the addition of crosslinking agents to the final stage, is useful for obtaining an appropriate manufacturing process to scale up to industrial scale and provide products with homogeneous quality.

The third liquid medium comprises one or more cross-linking agent (s) that are multifunctional, i.e., one or more cross-linking agent (s) having two or more reactive sites for forming covalent bonds to the hyaluronic acid molecule to be cross-linked. It is preferable that the cross-linking agent (s) used in the third step is bifunctional, that is, there are two reaction sites for forming a covalent bond to the hyaluronic acid molecule to be crosslinked. Useful multifunctional crosslinking agents include, but are not limited to, divinyl sulfone, multiepoxide and diepoxide such as 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethane di (EDDE) and die epoxyoctane, preferably BDDE. It is preferred that the at least one multifunctional crosslinking agent (s) provide ether crosslinking. The method advantageously provides an ether crosslinked hyaluronic acid gel product that is stable and readily sterile, e.g., autoclaved. Ester crosslinking is less stable and will be more easily hydrolyzed.

The crosslinking of the third method step is carried out under precipitation conditions in which a molded hyaluronic acid substrate is precipitated. In particular, the available surface of the hyaluronic acid molecule is present in a precipitated form due to precipitation conditions. The third liquid medium comprises one or more second organic solvent (s) in an amount to provide precipitation conditions for hyaluronic acid, and the amount and / or organic solvent (s) are selected such that the formed hyaluronic acid substrate The second liquid medium of the second method step for < / RTI >

As described above, it should be understood that the organic solvent is used to balance and transfer the solubility of hyaluronic acid during the manufacturing process. Those skilled in the art are well aware that other factors such as temperature, pH, ionic strength, and the type of organic solvent (s) may affect the concentration of organic solvent (s) that limit the precipitation of hyaluronic acid. The limiting concentration for precipitation of hyaluronic acid under given conditions is well known or can be readily ascertained by one of ordinary skill in the art.

Using this method, it is also possible to obtain a molded crosslinked hyaluronic acid product using a single crosslinking reaction in the third method step. Depending on the choice and number of crosslinkers in a single reaction step, the resulting molded product can be single crosslinked, i.e. it can contain essentially single type of crosslinking, preferably stable ether crosslinking, or can be multi-crosslinkable That is, it may contain two or more different types of bridges, preferably including stable ether bridges. It is surprising that processes using a single crosslinking reaction can achieve molded products with these desirable properties.

But are not limited to, organic solvents according to the present invention include but are not limited to pentane, hexane, cyclohexane, 1,4-dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, ethyl acetate, Diethyl ether, tetrahydrofuran, acetonitrile, methyl ethyl ketone, acetone, lower alkyl alcohols such as methanol, ethanol, propanol, isopropanol and butanol. The organic solvent according to the present invention is preferably water-soluble. A preferred group of organic solvents are lower alkyl alcohols. The term lower alkyl alcohol includes primary, secondary and tertiary alkyl alcohols having from 1 to 6 carbon atoms, i.e., C _1-6 alkyl alcohols. Specific examples of the lower alkyl alcohol include methanol, ethanol, modified distillation column, n-propanol, isopropanol, n-butanol, isobutanol and t-butanol. Preferred lower alkyl alcohols are methanol and ethanol, especially ethanol, due to their price, availability and ease of handling.

The third liquid medium of the third method step is suitably comprised as an aqueous medium, i.e. to some extent water. In addition to the crosslinking agent (s), the third liquid medium comprises 0-35 vol% of water and 65-100 vol% of the second water soluble organic solvent (s), preferably 20-35 vol% of water and the second water soluble organic solvent (s) 65 -80% by volume.

The third liquid medium has a pH of at least 11.5, i.e. the crosslinking is carried out at a pH of at least 11.5. As will be appreciated by those skilled in the art, the acidity of the hyaluronic acid starting material should be considered in order to obtain the desired pH in the third liquid medium. This can be achieved by addition of an acid, base, or buffer system with an appropriate buffering capacity. A preferred pH controlling agent is a strong base such as sodium hydroxide. The basic pH was found to be advantageous during crosslinking of precipitated hyaluronic acid molecules. The pH of the third liquid medium in the third step is preferably 13 or more. In a liquid medium comprising both water and the organic solvent (s), the measured pH may differ from the theoretical pH due to the type of solvent (s) and the amount of each solvent (s). Thus, the term apparent pH (pH _app ) is introduced under given conditions to refer to the pH measured using standard pH measurement equipment. The exact pH value can be readily ascertained, for example, by titration.

In the third method step, crosslinking produces a shaped crosslinked hyaluronic acid product according to the invention. The shaped crosslinked hyaluronic acid product according to the present invention is a gel or a hydrogel. That is, it can be considered to be water-insoluble, but when placed in a liquid, typically an aqueous liquid, it can be considered to be a substantially diluted crosslinking system of hyaluronic acid molecules. Since the crosslinking of the third method step is carried out under the precipitation conditions, both the molded hyaluronic acid substrate and the molded crosslinked product are precipitated. In particular, the available surfaces of both the shaped hyaluronic acid matrix and the shaped crosslinked product are present in a precipitated form due to the precipitation conditions of this third method step. The crosslinking reaction proceeds under appropriate conditions until the desired amount of crosslinking agent (s) reacts with the shaped hyaluronic acid substrate. The amount of crosslinker (s) bound to hyaluronic acid can be quantified and reported as the degree of modification (MoD), i.e., the molar amount of the combined crosslinker (s) relative to the total number of repeating HA disaccharide units. Until the degree of modification (MoD) of the crosslinked hyaluronic acid product is 1-40 units (0.1-4%) of crosslinker per 1000 units of disaccharide, preferably 1-10 units (0.1-1%) of crosslinker per 1000 units of disaccharide , It is preferable that the crosslinking reaction proceeds. The required reaction time depends on several factors such as hyaluronic acid concentration, cross-linker (s) concentration, temperature, pH, ionic strength and type of organic solvent (s). These factors are all well known to those skilled in the art and those skilled in the art will be able to adjust these related factors and other related factors thereby providing appropriate conditions to obtain a strain in the range of 0.1-4% The characteristics of the resulting product can be verified for the degree of deformation. The crosslinking in the third step is carried out for at least 2 hours, preferably at room temperature for at least 24 hours.

Any residual non-bond cross-linking agent (s) can be removed when the formed precipitated product is separated from the cross-linking medium. The shaped crosslinked hyaluronic acid product may be further purified by an additional rinsing step using a suitable rinsing liquid, for example, water, methanol, ethanol, saline or mixtures and / or combinations thereof.

Thus, the production process according to the invention enables the production of a predetermined physical form or structure of the crosslinked hyaluronic acid product, such as particles, fibers, strings, strands, nets, films, discs or beads. Longitudinal stretch: the ratio of the longest transverse elongation is at least 5: 1, such as at least 10: 1, such as at least 20: 1, optionally at least 100: 1 For example less than or equal to 25 000: 1, such as less than or equal to 100: 1, are particularly useful as medical implants or cosmetic implants because they can be dimensioned to prevent migration by having sufficient length, Because it can be easily administered by injection through a needle. For example, a crosslinked hyaluronic acid product according to the present invention having a single-stranded or fibrous shape filling a syringe of 20 mL or less has a thickness of 1 mm and a length of 25 m in its swollen state, i.e., its longitudinal elongation: The ratio of the longest transverse elongation thereof may be 25000: 1.

The predetermined structures may optionally be hollow or may comprise multiple layers. The space created in the predetermined structure of the hollow is optionally filled with HA, which may be crosslinked or substituted using a modification, such as another compound. One or more of the multiple layers in the predetermined layer structure can be composed of cross-linked HA or non-cross-linked HA, which can be chemically modified by substitution with other compounds. This can be achieved by arranging the hyaluronic acid substrate in a first method step, for example, by extrusion or molding, in a desired shape, or by extruding the dissolved hyaluronic acid substrate in a precipitation medium, for example ethanol, in a second method step . The obtained shape can be maintained throughout the manufacturing process and in the final product.

Optionally, the method further comprises a fourth step of treating the shaped precipitated, crosslinked hyaluronic acid product in non-precipitation conditions. That is, the method comprises four steps in a particular embodiment, or alternatively consists of four steps. This typically involves treating the shaped crosslinked hyaluronic acid product with a liquid medium and then returning it to the non-precipitated state. The liquid medium is typically water, saline or mixtures and / or combinations thereof, optionally containing an organic solvent, such as methanol or ethanol, in non-precipitation concentration. Due to crosslinking, the resulting formed hyaluronic acid product is a continuous network of interconnected and twisted hyaluronic acid chains that absorbs (swells) the liquid under non-precipitation conditions and forms a gel. The swelling can proceed until the gel is completely swollen and no more liquid can be absorbed or the swelling can be disturbed at a slightly earlier stage to obtain a partially swollen gel. The partially swollen gel may be useful as an intermediate product for further processing of the gel, for example, additional mechanical preparation of the gel structure with the desired size and shape may be performed. For example, the film may be cut into particles, slices, or pieces, the gel fibers may be cut into shorter pieces, and a well-defined irregular shape may be designed with a film or the like. The crosslinked HA fibers, strings or strands may also be woven together before or after drying, after drying, or after drying to form a net or film. It may also be convenient to use the partially swollen shaped gel product at the desired site during its implantation to facilitate administration, minimize patient discomfort, and enhance migration capacity by use of residual swelling capacity.

When the molded gel product undergoes non-precipitation conditions in an excess of liquid, its maximum swelling degree is confirmed, or conversely its minimum hyaluronic acid concentration (C _min ), that is, the hyaluronic acid It is possible to check the concentration. Using the manufacturing process according to the invention, it is possible to obtain a degree of swelling of 4-300 mL per gram of hyaluronic acid, preferably 15-180 mL per gram of hyaluronic acid. This is because the C _min value range is 0.3-25% (w / v), preferably 0.6-7% (w / v), the ranges are 3-250 mg / g, preferably 6-70 mg / . ≪ / RTI > While the desired degree of swelling (or C _min value) can be achieved with minimal deformation, it is very advantageous that the typical way of controlling the degree of swelling is by varying the degree of deformation. Thus, the present method provides a new concept for controlling the swelling capacity of the formed gel product, such adjustment is surprisingly hard molded gel is produced with in relation to help lower deformation of the gel uniquely high C _min values (low degree of swelling) .

The strain efficiency (MoE) is a measure of the ratio between the minimum HA concentration (C _min ) or stiffness / strength of the gel and the chemical strain by the cross-linking agent (s). Using the production process according to the invention it is possible to obtain a crosslinked hyaluronic acid product having a strain efficiency in the range of 10 or more, preferably 10-200, such as 20-150 or 20-190. Although not intending to be bound by any particular theory, the advantageous properties of the gel are the result of a very high degree of effective crosslinking, i. E. A large proportion of the combined crosslinking agent (s) (crosslinker ratios typically 0.35 or 35% (E.g., at least 40% or even at least 50%) is bound to hyaluronic acid at virtually two (or more) sites, with effective positioning of the bridges for the desired purpose, and possibly with an extremely high degree of kink As shown in Fig. Contrary to what one would expect from a low degree of modification of the hyaluronic acid product produced by the skilled artisan, the process according to the invention surprisingly provides a gel with high stiffness / strength. Under any circumstances, the method according to the present invention provides a useful way of further controlling the degree of swelling in relation to the strain. The process is also very suitable for continuous operation which is advantageous for large-scale production.

Optionally, the method also comprises a final step of separating the crosslinked hyaluronic acid product. That is, in certain embodiments, the method comprises four or five steps, or alternatively four or five steps. Depending on whether the product is kept under precipitation conditions or under non-precipitation conditions, this step may include separating the product into precipitated or non-precipitated forms. The separated, precipitated or non-precipitated product may then be sterilized to obtain a sterile, crosslinked hyaluronic acid product.

If desired, after the crosslinked hyaluronic acid product is obtained, other components such as local anesthetics (e.g., lidocaine hydrochloride), anti-inflammatory drugs, antibiotics and other suitable adjuvants, such as bone growth factors or bone- Can be added.

According to one aspect, the present invention provides a shaped crosslinked hyaluronic acid product. According to one embodiment, the product can be prepared or prepared by the process of the present invention. The shaped crosslinked hyaluronic acid product according to the present invention is a gel or a hydrogel. That is, it can be considered to be water-insoluble, but when placed in a liquid, typically an aqueous liquid, it can be considered to be a substantially diluted crosslinking system of hyaluronic acid molecules. The gel is generally liquid on a weight basis and may contain, for example, 90-99.9% water, but the gel behaves like a solid due to a three dimensional cross-linked hyaluronic acid network in the liquid. Due to its substantial liquid content, the shaped gel is structurally flexible and similar to natural tissue, which is very useful as a scaffold in tissue engineering and also useful for tissue augmentation. Along with the natural twist of the hyaluronic acid chain, it is the attachment site of the cross-linking agent and the hyaluronic acid molecule that imparts the properties and structure closely related to the swelling degree of the gel.

The amount of crosslinker (s) attached can be quantified and reported by the degree of modification (MoD), i.e. the molar amount of the combined crosslinker (s) relative to the total number of repeating HA disaccharide units. The degree of modification (MoD) of the crosslinked hyaluronic acid product according to the present invention is 1 to 40 units (0.1-4%) of crosslinker per 1000 units of disaccharide, preferably 1 to 10 units (0.1-1%) of crosslinker per 1000 units of disaccharide . The effect of the cross-linking reaction appears as the amount of attached cross-linker (s) attached at (at least) two ends to one (or more) hyaluronic acid chains and reported as the cross-linker ratio (CrR). The product according to the present invention preferably has a crosslinking agent ratio of at least 35%, preferably at least 40%, more preferably at least 50%, such as 35-80%, 40-80% and 50-80%. As a result, these products have a small number of crosslinking agents that do not provide effective crosslinking in the product. The high proportion of crosslinker provides very low total deformation with respect to gel strength, which again is advantageous to ensure high biocompatibility.

Another feature of the gel is its ability to absorb water until the gel is completely swollen. Adding more liquid will not further dilute the gel, i.e. the gel can not be infinitely diluted like a free molecule solution. When the gel undergoes non-precipitation conditions, it is also possible to confirm the degree of swelling of the gel, or conversely, its minimum concentration (C _min ), i.e. the concentration of hyaluronic acid when the gel product is completely swollen. The harder (less swellable) gel is generally less viscous than the softer (highly swellable) gel, more elastic, and is expected to have a longer half life in vivo. However, a harder gel can be recognized as foreign by the body if it is highly chemically modified. The product according to the invention preferably has a degree of swelling of 4-300 mL per gram of hyaluronic acid, preferably 15-180 mL per gram of hyaluronic acid. This means that the C _min value is in the range of 0.3-25% (w / w), i.e., 3-250 mg / g, such as 0.6-7% (w / w), i.e., 6-70 mg / g. The C _min value is preferably 0.6-5% (w / v), i.e., 6-50 mg / mL.

The shaped crosslinked hyaluronic acid product according to the present invention may be prepared by mixing one or more cross-linking agent (s), i.e., one or more cross-linking agent (s) having two or more reactive sites for forming covalent bonds with the hyaluronic acid molecule to be crosslinked It is preferable to be crosslinked. It is preferable that the cross-linking agent (s) is bifunctional, that is, there are two reaction sites for forming a covalent bond to the hyaluronic acid molecule to be crosslinked. Useful multifunctional crosslinking agents include, but are not limited to, divinyl sulfone, multiepoxide and diepoxide such as 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethane di (EDDE) and die epoxyoctane, preferably BDDE. The hyaluronic acid gel product is preferably crosslinked by stable ether crosslinking.

The shaped hyaluronic acid gel product may be multi-crosslinked, i.e. it may contain two or more different types of linkages, preferably including ether linkages. It is preferred that the hyaluronic acid product contains a single bridging, i.e. two or more different types of bridges, essentially including a single type of bridging, preferably an ether bridging. Single-bridged products have chemically well-defined advantages. It is advantageous for a product with a single type of cross-link to exhibit such desirable properties. In a specific embodiment, the hyaluronic acid product is crosslinked by ether crosslinking to provide a stable, autoclavable product.

While the desired degree of swelling (or C _min value) can be achieved with minimal deformation, it is very advantageous that the typical way of controlling the degree of swelling is by varying the degree of deformation. The main reason for minimizing deformation is to ensure that the gel has a high biocompatibility, but those skilled in the art are aware of other advantages as well. The molded product, with regard to assist deformation of the gel is characterized by a uniquely high C _min values (low degree of swelling). The strain efficiency (MoE) is a measure of the ratio between the minimum HA concentration (C _min ) reflecting the stiffness and strength of the gel and the chemical strain of the gel by the cross-linking agent (s). The shaped crosslinked hyaluronic acid product according to the present invention has a strain efficiency of 10 or more, preferably 10-200, such as 20-150 or 20-190. Products with a strain efficiency in the range of 10 or more, such as 20-190, are initially combined with low to moderate deformation, and at the same time combined with low swelling or medium swelling or liquid retention capacity. It is thus possible to provide a hard, crosslinked hyaluronic acid product which is biocompatible and has high deformation resistance.

Furthermore, it is preferred that the formed crosslinked hyaluronic acid gel product according to the present invention is viscoelastic. This means that the gel product exhibits a combination of viscosity and elasticity. As is well known to those skilled in the art, the viscoelastic properties can be ascertained with a rheometer. In the vibrational mode, the modulus of elasticity (G ') and the viscosity (G ") can be found at a frequency of 0.1 Hz or 1 Hz. In the case of certain viscoelastic gel products according to the invention,

The product according to the invention is optionally made up of a predetermined physical form or structure, such as particles, fibers, strings, strands, nets, films, discs or beads, including layers of different or different materials. Longitudinal stretch: the ratio of the longest transverse elongation is at least 5: 1, such as at least 10: 1, such as at least 20: 1, optionally at least 100: 1 For example less than or equal to 25 000: 1, such as less than or equal to 100: 1, are particularly useful as medical implants or cosmetic implants because they can be dimensioned to prevent migration by having sufficient length, Because it can be easily administered by injection through a needle. When fibers, strings or strands are formed, the ratio between their length and their width or their average diameter is at least 5: 1, such as at least 10: 1, such as at least 20: 1, alternatively at most 100: Such as 25 000: 1 or less, for example, 100: 1 or less. For example, a crosslinked hyaluronic acid product according to the present invention having a single-stranded or fibrous shape filling a syringe of 20 mL or less has a thickness of 1 mm and a length of 25 m in its swollen state, i.e., its longitudinal elongation: The ratio of the longest transverse elongation thereof may be 25000: 1. A preferred string thickness interval is 50-200 mu m.

The gel product may be designed as a hollow vessel containing human cells, drugs or other components. According to one embodiment of the present invention, the crosslinked HA gel product is useful as a drug delivery device and can be used in a drug delivery method. According to one embodiment of the present invention, the crosslinked HA gel product may be combined with non-crosslinked HA or crosslinked HA with different degrees of deformation or crosslinking. For example, the crosslinked gel product can be produced in the desired container shape. The container may form a depression of a different, e.g., lower, non-crosslinked HA or crosslinked HA, and then may be slowly released or contained to adjust the total strength of the resulting combined product Can be maintained.

Due to crosslinking, the shaped hyaluronic acid product is a continuous network of interconnected and twisted hyaluronic acid chains that absorbs (swells) the liquid under non-precipitation conditions and forms a gel. The swelling can proceed until the gel is completely swollen and no more liquid can be absorbed. Thus, the shaped crosslinked hyaluronic acid product can be provided in a fully swollen state. The swell may also be disturbed at a slightly earlier stage to obtain a partially swollen gel. The partially swollen gel may be useful as an intermediate product for further processing of the gel, for example, for the production of a gel structure or piece having a desired size and shape. It may also be convenient to use a partially swollen gel at the desired site during its implantation to facilitate administration and minimize patient discomfort. Thus, the shaped crosslinked hyaluronic acid product according to the present invention may also be provided in a partially swollen or non-swollen state.

The shaped crosslinked hyaluronic acid product according to the invention is useful for cosmetic surgery or medical surgery. Non-limiting examples of cosmetic surgery are skin rejuvenation and human contouring. Non-limiting examples of medical surgery include, but are not limited to, skin repercussion, human contouring, prevention of tissue adhesion, orthopedic application, incontinence treatment, treatment of vesicoureteral reflux (VUR) And forming a drainage channel to maintain the drainage channel. The shaped crosslinked hyaluronic acid product according to the present invention is also useful for drug delivery. Moreover, it can be used as a post-operative (intraperitoneal) attachment film, and can be used for hip and joint treatment.

According to one aspect, the present invention provides a method of treating a subject undergoing cosmetic surgery or medical surgery, comprising the step of administering a shaped crosslinked hyaluronic acid product according to the present invention to a subject in need thereof. Non-limiting examples of medical surgery include skin rejuvenation, human contouring, prevention of tissue adhesion, orthopedic applications, such as hip and joint treatment, and, for example, ophthalmology and drainage Channel formation.

According to one embodiment of the present invention, the shaped crosslinked hyaluronic acid gel product may be an additional structure or piece having different shapes of size greater than 0.1 mm when treated with a physiological salt solution. The crosslinked hyaluronic acid product according to the present invention has a longitudinally elongated shape and, in the fully swollen state, the longest transverse elongation is preferably less than 5 mm, for example less than 1.5 mm, preferably less than 0.2 mm. This is advantageous for injecting the swollen gel product through a syringe of the desired dimensions. In addition, the shaped crosslinked hyaluronic acid product according to the present invention has a longitudinally elongated shape and, in its fully swollen state, has a longitudinal elongation greater than 5 mm, preferably greater than 500 mm (0.5 m), such as 5 m Or even greater than 25 m. In particular, longitudinal stretching prevents migration and / or displacement of the grafted gel product in vivo.

The desired shape and size are arranged during manufacture of the product, i. E., By arranging the substrate into the desired shape prior to crosslinking. Another suitable way of obtaining the desired structure size is to prepare the shaped cross-linked hyaluronic acid gel at the desired concentration and then subject the gel to mechanical disturbance, such as mincing, mashing, Passing the swollen gel or precipitated crosslinked product through a filter or mesh having an appropriate pore size. The resulting gel particles or pieces are dispersed in a physiological saline solution to obtain a gel dispersion or slurry having particles of the desired size and shape. Depending on the shape, the size of the gel structure can be ascertained by any suitable method, such as laser diffraction, microscopy, filtration, etc., and is determined by the longest distance between the two ends of the particle. For spherical structures, the diameter is equal to the size for this purpose.

The useful gel structure size range and shape will depend on the intended application. Gel particles, pieces or fibers of greater than 0.1 mm in size are useful when treated with a physiological saline solution, in soft tissue augmentation, preferably subcutaneous, intramuscular or supraperiostal administration. As used herein, the term "soft tissue augmentation " refers to an increase in the volume of any type of soft tissue, including, but not limited to, facial contours (e.g., more prominent cheeks or jaws), correction of concave deformities Dysmenorrhoea), and correction of deep facial wrinkles associated with aging. Thus, soft tissue augmentation can be used for cosmetic or medical purposes, for example, after trauma or degenerative diseases. These two objectives are easily distinguished by those skilled in the art. As used herein, the term "soft tissue" refers to a tissue that connects, supports, or surrounds other organs of the body. Soft tissues include muscle, fibrous tissue and fat. Soft tissue augmentation can be performed in any animal, including humans. The method is preferably carried out in humans.

The term "subepidermal administration" or "subcuticular administration" as used herein refers to administration beneath the skin epidermis and refers to subcutaneous administration of the dermis, subcutaneous tissue or even deeper, Into the applicable periosteum (around the bone tissue).

Administration of the gel structure can be performed in any suitable manner, such as injection from a needle of a standard cannula and an appropriate size, or surgical insertion, for example, in the case of film administration. Administration is performed where soft tissue augmentation is desired, e.g., on the chin, cheek or face or anywhere in the body.

Implants according to the present invention may be used in combination with a shaped crosslinked hyaluronic acid product according to the invention, for example a hyaluronic acid gel structure having a size of 0.1 mm or more, such as particles, beads, fibers or cut strips, Composition. The compositions typically comprise a physiological salt buffer. The composition may further comprise other suitable additives such as local anesthetics (e.g. lidocaine hydrochloride), anti-inflammatory drugs, antibiotics and other suitable adjuvants such as bone growth factors or bone growth cells. The shaped crosslinked hyaluronic acid product or an aqueous composition thereof according to the present invention is provided in a pre-filled syringe, i.e., a pre-filled syringe with a sterilized molded cross-linked hyaluronic acid product or a molded product comprising the molded product . Alternatively, the shaped crosslinked hyaluronic acid product may be maintained in a precipitated form in a syringe, bag, or other suitable container, and may be in its non-precipitated form in the body before or after injection.

The swollen or partially swollen, shaped crosslinked hyaluronic acid product is preferably autoclavable, since the autoclave is the most convenient way to sterilize the final product. Thereby, a sterilized, molded crosslinked hyaluronic acid product can be produced.

The size of the gel structure, e. G., The fiber according to the invention, of course, depends on the extent to which the gel was swollen and the ionic strength of the buffer, solution or carrier contained in and / or surrounding the gel structure. Throughout this specification, a given structure size assumes physiological conditions, in particular isotropic conditions. While it is desirable for the gel structure to contain and disperse a physiological salt solution in the solution, the gel structure according to the invention can be of different sizes by treating the gel structure with another tonicity, different pH solution It should be noted that what is supposed to be or the gel structure could not be swollen to its maximum size.

Herein, the physiological or isotonic solution is a solution having an osmolarity of 200-400 mOsm / l, preferably 250-350 mOsm / l, more preferably about 300 mOsm / l. In fact, such osmolarity is easily achieved by the preparation of a 0.9% (0.154 M) NaCl solution.

The shaped crosslinked hyaluronic acid gel product according to the invention is stable but not permanent under physiological conditions. Stability in vitro is mentioned by a stability study accelerated for 14 days at 60 < 0 > C in Example 7. In one embodiment according to the present invention, at least 70%, preferably at least 90% of the formed crosslinked hyaluronic acid gel product is maintained in the test tube for at least 2 weeks, more preferably 2 weeks to 2 years. The term "degraded" means that less than 20%, preferably less than 10%, of the medium remains in the body.

The shaped crosslinked hyaluronic acid gel product according to the present invention is more resistant to degradation than natural hyaluronic acid in vivo. The presence of the elongated stable gel product is beneficial to the patient because the time between treatments is prolonged. In addition, the product is very similar to natural hyaluronic acid in order to maintain high biocompatibility of natural hyaluronic acid. Products similar to native HA should be degraded by the hyaluronidase enzyme, preferably at least 99%. The biodegradability of the rigid hyaluronic acid gel product according to the present invention is referred to in Example 8, wherein the product is recognized by hyaluronidase. This reflects the similarity of the product with natural hyaluronic acid.

Justice

Throughout this disclosure, the following terms are defined as follows.

Terms characteristic meaning HA HA refers to sodium hyaluronate Gel-type HA Gel-form HA is a cross-linked HA that can not be extracted from the gel by, for example, rinsing with saline, in contrast to extractable HA. Extractable HA The extractable HA is a cross-linked HA or non-cross-linked HA that can be extracted, for example, by rinsing with saline. C _HA HA concentration

mg/g, mg/mL, %(w/w), %(w/v)으로 표현된다.

mg / g, mg / mL,% (w / w) and% (w / v)
SwD Swelling degree

SwD is preferably expressed in g / g, mL / g or dimensionless numbers. SwD is the reciprocal of the concentration of gel-form HA in the gel that is fully swollen in 0.9% saline, i.e., the volume or mass of the fully swollen gel that can be formed per gram of dry cross-linked HA. SwD describes the maximum liquid-absorbing (0.9% saline) capacity of the product.
C _min Minimum HA concentration The concentration of gel-like HA in gels that are completely swollen in 0.9% saline, expressed as mg / g or mg / mL.

GelC Gel content

g / g, an irregular number or%. The gel content is the ratio of HA bound in gel-form to the total HA content in the product. MoD Deformation diagram

Mole / mole, an irregular number, or a mole%. MoD describes the amount of cross-linking agent (s) bound to the HA, i.e., the molar amount of the combined cross-linker (s) to the total molar amount of repeating HA disaccharide units. MoD reflects the extent to which the HA is chemically modified by the cross-linking agent (s). CrR Ratio of crosslinking agent

Here, X is a crosslinking agent. CrR can also be expressed as: < RTI ID = 0.0 >

Mole / mole, an irregular number, or a mole%. CrR describes the ratio of total bound crosslinkers (HA-X-HA and HA-X) bound to two (or more) disaccharides (HA-X-HA only). MoE Strain efficiency

MoE is an irregular number obtained as a ratio between C _min expressed in mg / g and Mo D expressed in%. MoE describes the amount of interactions caused by both chemical modification and molecular twist achieved at a particular rate of chemical modification by the cross-linking agent (s).
Example: For a product with C _min = 35 mg / mL and MoD = 1.15%, the MoE is approximately 30 and is calculated as follows:

G ' Elastic modulus The elastic modulus describes the resistance of the gel to elastic deformation and is expressed in Pa (Pascal). Strong gels will provide a larger number compared to weaker gels. G '' Viscosity ratio The viscosity rate describes the resistance of the gel to viscous strain and is expressed in Pa (Pascal) units. In addition to G ', the viscosity rate describes the total resistance to deformation.

Without intending to be limited thereto, the present invention will be illustrated in the following examples.

Example  sector

Analysis method

Identification of HA concentration

The method of confirming the HA content is described in Ph. Eur. 1472 < / RTI > from the analytical test for sodium hyaluronate. The principle of this method is that the condensation reaction of the furfural derivative formed by heating in sulfuric acid is carried out using a carbazole reagent to form a purple product. This reaction is specific to the D-glucuronic acid moiety of HA. Absorbance is measured at 530 nm and glucuronic acid is used for standardization.

The product formed from the content of D-glucuronic acid (GlcA) in the sample is confirmed by reaction with carbazole. To obtain a homogeneous sample solution, the stabilized gel of HA is digested with sulfuric acid at 70 ° C and diluted with 0.9% NaCl-solution. The solution is mixed with sulfuric acid at 95 占 폚 and then mixed with the carbazole reagent. A purple solution is obtained by the reaction. The intensity of the color is measured with a colorimeter at 530 nm and the absorbance of each sample is directly proportional to the GlcA- content. HA content is calculated from the GlcA- content in each sample.

Gel content ( GelC ) Confirmation of

GelC describes the percentage of total HA bound in gel form in%. The gel content is defined as the amount of HA in the sample that did not pass through the 0.22 占 퐉 filter. GelC is calculated from the amount of HA collected in the filtrate, the extractable HA referred to herein. Gel content and extractable HA content are provided as a percentage of the total amount of HA in the gel sample. Briefly, the gel content is confirmed by mixing a specific amount of gel sample with 0.9% NaCl in a test tube. The gel is allowed to swell and then the NaCl-phase is separated from the gel-phase by filtration through a 0.22 [mu] m filter. The concentration of HA in the filtrate is checked by confirming the HA concentration.

Swelling degree ( SwD ) Confirmation of

SwD describes the liquid-absorbing power of the product, i. E., The ability to absorb 0.9% NaCl. The product is typically a dry, crosslinked HA gel. The dried product can be precipitated or can not be precipitated. SwD can be ascertained from the weight of the fully swollen product formed when swelling a specific weight dry product in saline. If the dry product precipitates, it will return to the non-precipitated form under these conditions.

The dry product of the predetermined weight (typically 0.1 g) is treated with an excess of 0.9% NaCl (aq) and the product is allowed to swell at room temperature (23 ° C) for 1 hour. After removing the non-absorbed liquid, the fully swollen product is collected and weighed. Removal of the non-absorbed liquid also removes any components that are not bound or kinked in the cross-linked gel, such as non-cross-linked hyaluronate molecules and lightly cross-linked hyaluronate molecules. Since they will not contribute to the weight of the fully swollen product and thereby provide a seemingly lower SwD, a correction to the gel content must be made to identify the true SwD. If the gel content is believed to be close enough to 100% that the result is unaffected, such correction may be omitted.

SwD is calculated as the ratio between the weight of the product that is completely swollen and rinsed from the extractable HA as described above and the weight of the dry crosslinked HA in the product:

,

,

Here, GelC is expressed as an irregular number. If GelC is considered to be close to 1 (100%), SwD can be calculated as:

Three techniques were used to collect the completely swollen product and to remove the non-absorbed liquid: 1) a technique of collecting the swollen product in pieces and contacting the product with a dry surface, 2) using a metal net, A technique of collecting all of the swollen product and contacting the net with the dry surface, and 3) a technique of removing liquid from the swelled product by inhalation through a 0.2 [mu] m filter. In the latter case, the weight of the fully swollen product was not precisely weighed but was calculated from the weight of the dry sample before swelling, the weight of the liquid added, and the weight of liquid removed during filtration using the following equation:

m _{Completely Swollen  Gel product}  = m _{Added  Liquid}  - m _{Removed  Liquid}  + m _{dry  product} .

Notably, a stronger gel would have a lower SwD, while a weaker gel would have a higher SwD.

Minimum concentration ( C _min ) Confirmation of

C _min describes the concentration of gel-like HA in the crosslinked HA gel product completely swollen at 0.9% NaCl after removing all extractable HA. Since the product is no longer able to absorb liquid, this concentration is the minimum HA concentration that can be obtained for this particular gel product. Notably, the stronger gel will have a higher C _min , while the weaker gel will have a lower C _min .

C _min is confirmed similar to the confirmation of SwD as described above, using the following relationship:

.

.

The degree of deformation (MoD)  Confirm

MoD describes the molar amount of bound crosslinker (s) to the total number of repeating HA disaccharide units. This measurement does not distinguish between the mono-linked cross-linker (s) and the actually cross-linked cross-linker (s), i.e., all cross-linker (s) bound to the HA via one or more covalent bonds. For example, for HA gels crosslinked with BDDE, 1% MoD means that there is one bound (mono linked or crosslinked) molecule of BDDE per 100 disaccharide units in the HA gel.

MoD is confirmed using NMR spectroscopy on enzymatically degraded gel products. Soluble HA, residual (non-bonded) cross-linker (s) and derivatives thereof are cleaned and removed prior to gel breakdown by filtration over a 0.22 탆 filter. Gel product in Aspergillus at 37 ℃ bakteo brother LES sense (Arthrobacter The enzyme is degraded by enzymatic treatment with Conradoinase AC derived from aurescens . The degraded gel product is subjected to NMR spectroscopy by recording a one-dimensional ¹ H NMR spectrum on a 400 MHz spectrometer equipped with a standard 5 mm probe.

The NMR spectrum shows the signal at δ _H 1.6 ppm originating from 4 protons in the coupled BDDE molecule and the signal at δ _H 2.0 ppm originating from 3 protons of the CH ₃ group on the HA disaccharide N-acetylglucosamine residue . The ratio between the integral values for these two signals is proportional to the ratio between the molar amount of the bound BDDE and the molar amount of the disaccharide after correction for the signal of the proton involved in each signal, and MoD is obtained.

Strain efficiency MoE ) Confirmation of

MoE is the ratio between the minimum HA concentration and the deformation of the gel, i.e.,

to be.

C _min (mg / g or mg / mL) and MoD (%) are identified as described above. Since C _min is closely related to the gel strength, MoE is a measure of how efficient the crosslinking procedure is in producing the gel of the desired strength. A high MoE process will produce gels with high C _min and low MoD, that is, despite the limited chemical modification of HA, strong gels are produced.

Cross-linking agent  ratio( CrR ) Confirmation of

CrR describes the ratio of total bound cross-linker (s) (HA-X-HA and HA-X) combined with two (or more) disaccharides (HA-X-HA)

Here, X is a crosslinking agent.

The method as asbestos bakteo Conde's brother Roy Les Senses derived proteinase AC or Proteus vulgaris (Proteus vulgaris) decomposing the HA gel using corned Roy proteinase derived from the AB, the fragment (X-HA) with a cross-linking agent bound to a fragment consisting of key disaccharide (△ di-HA), and containing 1-16 of disaccharides , And then based on confirmation of HA-X fragments using SEC-UV-MS. Fragments are separated by size exclusion chromatography (SEC) and detected using mass spectrometry (MS). The peak areas for each fragment group are summed and CrR is calculated as follows:

.

.

It is assumed that all types of HA-X fragments have the same reaction in the MS detector, i.e., the specific peak region corresponds to the given molar amount for all types of HA-X fragments (Kenne et al. , Carbohydrate Polymers 91 (2013) 410-418).

When identifying CrR, care must be taken only to include BDDE bound by ether linkage. Depending on the conditions during the crosslinking, the BDDE can be bound to HA via both ether linkages and ester linkages. Since the ester linkages are easily hydrolyzed, it is only the ether-bound BDDE that contributes to gel strength and long-term storage. Fragments with ester-bound BDDE have the same mass as ether-bound BDDE but can be detected because they have slightly different chromatographic retention times. To identify CrR without any ester-bound BDDE, the sample must be hydrolyzed prior to analysis. Hydrolysis of the sample can be carried out, for example, either before or after enzymatic degradation by adding a base and / or applying heat.

Identification of pH

pH verification is performed potentiometrically at ambient temperature using a glass electrode. The method used to determine the pH of the swollen product is based on the USP method <791>. Procedure: Calibrate the pH-meter with buffer for normalization at ambient temperature, pH 7.0 and pH 4.0. (For all measurements) Transfer approximately 1.2 mL of sample to an appropriate container. Ensure that the sample is at room temperature. Between each measurement, the electrode is rinsed in distilled water and carefully wiped off.

The method for the pH determination of the process solution, particularly the organic solvent, is the same as above, but the correction is carried out at pH 7.0 and pH 10.0.

The pH value measured in a mixture of water and the organic solvent (s) differs in comparison to the pH measured in a pure water solution for the same concentration of base, for example sodium hydroxide (NaOH). Thus, the expression pH (pH _app ) is used for the pH measured in an aqueous mixture of the organic solvent (s). The apparent pH depends on several factors, the type of organic solvent (s), the concentration of the organic solvent (s), the temperature, the ionic strength and the presence of other compounds in the mixture.

The apparent pH values reported in the experimental section were measured with a Mettler Toledo MA 234 pH / Ion analyzer using a Mettler Toledo Inlab Routine Pro electrode for pH range 0-14.

Flow measurement

Vibrational flow measurements were used to confirm the viscoelasticity of the swollen gel product. The elastic modulus (G ') describes the gel strength in terms of the physical resistance of the gel to elastic deformation. The viscosity ratio (G &quot;) describes the gel strength in terms of the physical resistance of the gel to viscous deformation. The measurement is carried out using a vibrating flow meter.

The flow measurement is performed as follows. A frequency sweep is made with a relaxation time of at least 15 minutes between sample loading and measurement with a strain (γ) of 0.1%. Use a parallel plate probe with a diameter of 25 mm at 2 mm intervals. The average value of the modulus of elasticity (G ') and the viscosity (G ") is evaluated at 0.1 Hz and 1 Hz from the frequency sweep. To demonstrate that a frequency sweep was performed on the strain (y) within the linear viscoelastic range, an amplitude sweep was made at 1 Hz.

enzyme Degradable  Confirm

To demonstrate that the crosslinked gel is the same as native HA in terms of biodegradability, enzymatic degradation is performed and it is verified that it is degraded by hyaluronidase from a positive testis.

A predetermined weight of product (dried or swollen to a known concentration) is swollen in 100 mL of 0.9% saline at 37 DEG C overnight. Amniotic testis-derived hyaluronidase (Type II, H2126 Sigma) is added to obtain an enzyme concentration of 200 U / mL and the preparation is set to shake overnight at 37 ° C. The solution is filtered through a 0.22 [mu] m filter and the amount of HA in the filtrate is verified using the carbazole method. Decomposition is calculated as the ratio of the amount of HA in the filtrate: the total amount of HA in the sample.

Example

Example 1 - Crosslinking Process

About 1 g of sodium hyaluronate with a molecular weight of 0.8-3 MDa was dissolved in 10-50 mL of an aqueous solution containing 0-40% ethanol and 0-120 mM strong base. After complete dissolution, the resulting hyaluronate solution had a pH _app of 6-13 as measured as previously described. (Manufacturing step)

The hyaluronate solution was extruded through a 0.3-0.5 mm diameter opening into a liquid medium containing an organic solvent. The extruded hyaluronate solution immediately precipitated into hyaluronate fibers. (Precipitation step)

Approximately 1 g of precipitated HA fiber is then mixed with about 20-70 mL of an aqueous crosslinking medium comprising 65-80% organic solvent, 0.3-1.2 g (30-230 mM) of a crosslinking agent, and 1-75 mM strong base . Hyaluronate precipitated and remained in the form of fibers. The crosslinked medium containing HA produced had a pH of 11.5 or higher. The HA fibers were allowed to crosslink in an aqueous / organic crosslinking medium. (Crosslinking step)

The crosslinked hyaluronate fibers thus obtained were removed from the crosslinking medium and neutralized with phosphoric acid or the like. The crosslinked HA fibers were dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 1 hour.

The precipitated intermediate product is characterized as follows. The skewness of dry crosslinked hyaluronate fibers was measured. The dried fibers were soaked in an excess of 0.9% aqueous saline to allow the liquid to fully absorb. The fibers then formed an aqueous gel and allowed to absorb the saline solution until equilibrium (maximum) swelling of the fibers was achieved. The gel fibers were removed manually from a solution of excess saline and non-crosslinked hyaluronate and transferred directly to the scale. The weight of the sufficiently swollen fibers was measured. From the saline water absorption results, the degree of swelling (SwD) corresponding to a specific sodium hyaluronate concentration (C _min ) in the sufficiently swollen gel was measured.

The degree of modification (MoD) of the resulting crosslinked hyaluronate fibers was determined by NMR spectroscopy as described above. Briefly, the intensity of the signal from the linked BDDE is related to the signal from the acetyl group in the HA. The molar ratio of bound BDDE-related HA can be calculated after correcting the number of protons that cause the signal. Corresponding strain efficiency (MoE) was calculated from swelling degree.

The precipitated intermediate product of the crosslinked HA fibers described above was allowed to swell to the stated HA-concentration as follows. Phosphate buffered saline solution (4 mM phosphate and 150 mM NaCl) was added to the dried HA fibers at a concentration of about 10-40 mg HA / mL. The partially swollen gel was filled into a 10 mL plastic syringe. The filled syringes were sterilized using moist heat at 123 캜 for a final equilibration time (F ₀ ) of about 20 minutes. The content of autoclaved syringes was characterized in terms of pH, HA concentration, swelling degree (SwD) and minimum concentration (C _min ) in the manner described above, and MoE was calculated from the MoD determined for the intermediate product.

Crosslinked HA fibers were prepared as described above. The analytical data for the precipitated intermediate product are shown in Table 1 below and the data obtained for the swollen and autoclaved product are shown in Table 2 below.

Product Characterization  (Precipitated intermediate product) Experiment Id Dry HA amount (mg) Amount of swollen HA (mg) SwD *
(g / g) C _min (mg / mL) MoD (%) MoE One - - - - 0.3 - 2 23 1181 51 20 0.7 28 3 15 2200 147 7 0.2 38 4 17 1070 62 16 0.2 73 5 17 1505 89 11 0.4 30 6 16 2110 133 8 0.2 42 7 - - - - 1.3 - 8 - - - - 0.4 - 9 - - - - 0.5 - 10 - - - - 0.2 - 11 - - - - 0.3 - 12 16 501 32 31 1.7 19 13 15 313 21 48 0.3 190 14 19.8 632 32 31 0.7 46 15 - - - - 0.5 - * Measured according to process 1

Characterization of swelled and autoclaved products Experiment Id pH HA concentration (mg / mL) SwD **
(mL / g) C _min
(mg / mL) MoE One 6.9 20 * 110 9 28 2 - - - - - 3 7.3 19 85 12 65 4 - 20 * - - - 5 - - - - - 6 *** 7.2 20 * 139 7 40 7 7.6 20 * 23 43 32 8 6.6 20 * 40 25 59 9 7.1 20 * 21 47 97 10 7.2 20 * 96 10 44 11 7.3 20 * 55 18 55 12 - - - - - 13 7.3 20 * 23 44 174 14 - - - - - 15 7.9 20 * 97 10 21 * Estimated HA concentration ** Measured according to Step 3 *** In the swelling step described above, 2 mg lidocaine hydrochloride per mL of phosphate buffered saline solution was added

Example  2 - Different As a crosslinking agent  Bridging

A hyaluronate solution was prepared as described in Example 1. After complete dissolution, the resulting hyaluronate solution had a pH of 9 or higher when measured as previously described. The hyaluronate solution was extruded through a 0.5 mm diameter opening into a liquid medium containing 99.5% ethanol. The extruded hyaluronate solution immediately precipitated into hyaluronate fibers.

Approximately 1 g of precipitated HA fibers were transferred to an aqueous / organic crosslinking medium comprising 70% ethanol, a crosslinking agent and NaOH. Hyaluronate precipitated and remained in the form of fibers. The crosslinked medium containing HA produced had a pH of 11.5 or higher.

The HA fibers were allowed to crosslink in an aqueous / organic crosslinking medium. The resulting crosslinked hyaluronate fibers were then removed from the crosslinking medium and neutralized with phosphoric acid. The crosslinked HA fibers were dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 2 hours.

The precipitated intermediate product of the crosslinked HA fibers described above was allowed to swell to the stated HA-concentration as follows. Phosphate buffered saline solution was added to the dried HA fibers at a concentration of approximately 20 mg HA / mL. The partially swollen gel was filled into a 10 mL plastic syringe. The filled syringes were sterilized using moist heat at 123 캜 for a final equilibration time (F ₀ ) of about 20 minutes. The contents of the autoclaved syringes were characterized in terms of pH, swelling degree (SwD step 3), minimum concentration (C _min ), strain (MoD) and strain efficiency (MoE) in the manner described above. The results shown in Table 3 suggest that the different die epoxide is a useful cross-linker in the process.

Characterization of the product Bridging pH SwD * (mL / g) C _min (mg / mL) MoD (%) MoE EDDE ** 7.6 24 41 1.5 27 EDDE ** 7.3 12 86 2.8 31 Die epoxyoctane 7.9 97 10 0.5 21 * Measured according to Step 3, HA concentration estimated to be 20 mg / mL ** 1,2-ethanediol diglycidyl ether, purity 50%

Example  3 - Crosslinking in methanol

A hyaluronate solution was prepared as described in Example 1. After complete dissolution, the resulting hyaluronate solution had a pH of 9 or higher when measured as previously described. The hyaluronate solution was extruded through a 0.5 mm diameter opening into a liquid medium containing 99.5% ethanol. The extruded hyaluronate solution immediately precipitated into hyaluronate fibers.

Approximately 0.75 g of precipitated HA fibers were transferred to an aqueous crosslinking medium comprising 80% methanol, 1,4-butanediol diglycidyl ether (BDDE), and NaOH. Hyaluronate precipitated and remained in the form of fibers. The crosslinked medium containing HA produced had a pH of 11.5 or higher.

The HA fibers were allowed to crosslink in an aqueous / organic crosslinking medium. The resulting crosslinked hyaluronate fibers were then removed from the crosslinking medium and neutralized with phosphoric acid. The crosslinked HA fibers were dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 1 hour.

The degree of swelling was measured according to SwD step 1. The dried intermediate product of the crosslinked fibers was allowed to swell to 20 mg HA / mL in phosphate buffered saline solution. The swollen gel was filled into a 10 mL plastic syringe. The filled syringes were sterilized using moist heat at 123 캜 for a final equilibration time (F ₀ ) of about 20 minutes. The content of autoclaved syringes was characterized in terms of pH and strain (MoD) in the manner described above. The resulting gel formulation had a pH of 6.8 and an MoD of 0.34%.

Example 4 - Crosslinking in isopropanol

A hyaluronate solution was prepared as described in Example 3. Approximately 0.5 g of precipitated HA fiber was transferred to 30 mL of an aqueous crosslinking medium containing 70% isopropanol, 1,4-butanediol diglycidyl ether (BDDE), and NaOH. Hyaluronate precipitated and remained in the form of fibers. The crosslinked medium containing HA produced had a pH of 11.5 or higher. The crosslinked medium containing HA produced had a pH of 11.5 or higher.

The HA fibers were allowed to crosslink in an aqueous / organic crosslinking medium. The resulting crosslinked hyaluronate fibers were then removed from the crosslinking medium and neutralized with phosphoric acid. The crosslinked HA fibers were dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 1 hour.

The dried intermediate product of the crosslinked fibers was allowed to swell to a HA-concentration of 20 mg HA / mL in phosphate buffered saline solution. The swollen gel was filled into a 10 mL plastic syringe. The filled syringes were sterilized using moist heat at 123 캜 for a final equilibration time (F ₀ ) of about 20 minutes. The contents of the autoclaved syringes were characterized in terms of pH, degree of swelling (according to Process 3), gel content and degree of deformation (MoD) in the manner described above. The minimum concentration C _min and strain efficiency (MoE) were calculated as described above. The resulting gel formulation had a pH 7.6, SwD 47 g / g HA, C min 21 mg / mL, MoD 0.41%, and MoE 52.

Example 5 - Cross-linking to a desired shape and structure

A hyaluronate solution was prepared as described in Example 1. After complete dissolution, the resulting hyaluronate solution had a pH of 9 or higher when measured as previously described.

A precipitated HA substrate with different morphology or structure was prepared from the hyaluronate solution in the following manner:

1) extrusion through a 0.5 mm diameter opening forming a droplet;

2) extrusion through a 0.5 mm diameter opening forming the net;

3) spreading on a plastic foil forming the film, and

And then immersed in a liquid medium of 99.5% ethanol. The extruded hyaluronate solutions were immediately precipitated into hyaluronate droplets, net and film, respectively.

Thereafter, about 1 g of each deposited HA substrate was transferred to 45 mL of an aqueous crosslinking medium comprising 70% ethanol, 1,4-butanediol diglycidyl ether (BDDE) and NaOH. During the crosslinking reaction, the hyaluronate precipitated and remained in the predetermined form. The crosslinked medium containing HA produced had a pH of 11.5 or higher.

After crosslinking, the crosslinked hyaluronate formulation was manually removed from the crosslinking medium, neutralized with phosphoric acid, and dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 1 hour before characterization of the formulation.

The degree of swelling (SwD) was measured for droplets and film. A known amount of dried cross-linked hyaluronate sample was immersed in an excess of 0.9% aqueous saline to allow the liquid to fully absorb. The sample then formed an aqueous gel and absorbed the saline solution until equilibrium (maximum) swelling of the sample was achieved. The gel sample was then passively removed from a solution of excess saline and finally non-crosslinked residual hyaluronate and transferred directly to the scale. The maximum saline uptake was estimated by comparing the weight of the swollen gel to the initial weight of the dry material. The results are presented in Table 4 as both the degree of swelling (SwD step 1) and the minimum concentration (C _min ). The nets were swollen in an excess of 0.9% aqueous saline as described above. The swollen gel is shown in Fig.

The cross-linked hyaluronate droplets and the strain (MoD) of the film were confirmed by NMR spectroscopy as described above. Briefly, the signal intensity from the connected BDDE is related to the signal from the acetyl group in HA. Then, after correcting for the number of signaling protons, the molar ratio of bound BDDE to HA was calculated. The results of MoD and calculated strain efficiency (MoE) are presented in Table 4.

The dried intermediate product of the cross-linked droplets was swollen in phosphate buffered saline to a HA concentration of 20 mg HA / mL. The swollen gel was filled into a 10 mL plastic syringe. The filled syringe was sterilized by moist heating at 123 ° C, where F ₀ was about 20 minutes. The results from the autoclaved droplets are also presented in Table 4.

Table 4 Sample SwD (g / g HA) C _min (mg / mL) MoD (%) MoE Droplet 60 * 17 0.47 36 Autoclaved droplet 58 ** 17 0.47 37 film 115 * 9 0.43 20 * Measured according to process 1 ** Measured according to Step 3

The results show that it is possible to produce a pre-determined type of crosslinked HA material that is retained during the crosslinking process and autoclave.

Example  6 - Crosslinking of alkaline fibers

A hyaluronate solution was prepared as described in Example 1. After completely dissolving, the hyaluronate solution produced had a pH of greater than 9 when measured as above. This hyaluronate solution was pushed through a 21G needle (0.5 mm in internal diameter) into a 99.5% ethanol liquid medium. The extruded hyaluronate solution immediately precipitated into hyaluronate fibers.

Approximately 1 g of the precipitated non-washed HA fiber was transferred to 65 mL of a crosslinked aqueous solution containing 70% ethanol and 1,4-butanediol diglycidyl ether (BDDE). The hyaluronate was still in a precipitated state, retaining its basic and fibrous form. The crosslinked medium obtained after the addition of the basic HA fibers had a pH higher than 11.5.

The HA fibers were crosslinked in an aqueous / organic crosslinking medium. The resulting crosslinked hyaluronate fibers are then taken from the crosslinked aqueous medium and neutralized by the addition of phosphoric acid and dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 1 hour, Respectively.

The crosslinked fiber-dried intermediate product was swollen at a HA-concentration of about 20 mg / mL in a phosphate buffered saline solution. A somewhat swollen gel was filled into a 10 mL plastic syringe. The filled syringes were sterilized by wet heat treatment at 123 ° C and the final F ₀ was approximately 20 minutes. The pH, swelling degree (SwD), gel content and degree of modification (MoD) of the contents of the autoclaved syringe were determined according to the method described above. The minimum concentration C _min and modification efficiency (MoE) were calculated as described above. The results are shown in Table 5.

Formulation The pH of the gel formulation SwD * (g / g HA) C _min
(mg / mL) MoD (%) MoE One 6.6 170 6 0.10 59 2 6.8 51 20 0.25 78 * Measured according to Procedure 3 and calibrated with gel content

That is, through the crosslinking of the basic fibers in a neutral crosslinked medium containing 70% alcohol having a final approximate pH of 11.5 higher than the above, fully formed gel fibers were obtained.

Example 7 - Biocompatibility and stability experiment

Four formulations were prepared as described in Example 1 for the purpose of investigating the biocompatibility of the materials in vivo. After completely dissolving, the hyaluronate solution produced had a pH of greater than 9 when measured as above. The hyaluronate solution was pushed through 99.5% ethanol liquid medium through openings of 18G (Phi 0.8 mm; Formulations 1 and 2) or 25G (Phi 0.3 mm; Formulations 3 and 4) size. The extruded hyaluronate solution immediately precipitated into hyaluronate fibers.

Approximately 1 g of the precipitated non-washed HA fibers were transferred to 60 mL of a crosslinked aqueous solution containing 70% ethanol, 1,4-butanediol diglycidyl ether (BDDE) and NaOH. Formulations 2 and 4 used BDDE concentrations four times higher than Formulations 1 and 3. The hyaluronate was still in a precipitated state and retained its fibrous morphology. The crosslinking media obtained with HA had a pH greater than 11.5 as measured by the apparatus described above.

The HA fibers were crosslinked in an aqueous / organic crosslinking medium. The resulting crosslinked hyaluronate fibers are then taken from the crosslinked aqueous medium and neutralized by the addition of phosphoric acid and dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 1 hour, Respectively.

The dried fibers were placed in an excess of 0.9% saline to allow them to absorb liquid freely. Afterwards, the fibers became aqueous gels and absorbed saline until equilibrium (maximum) swelling of the fibers was reached. The gel fibers were then manually removed from excess saline and non-crosslinked hyaluronate solution and immediately transferred to a scale. The weight of the fully swollen fibers was compared to the initial weight of the precipitated material to calculate the maximum absorption of saline. The results obtained from the gel fibers before the sterilization treatment are shown in Table 6 as swelling degree (SwD) and HA minimum concentration (C _min ).

Formulation Weight of dry fiber (mg) Weight of fully swollen fiber (mg) SwD * (g / g HA) C _min (mg HA / mL) MoD (%) CrR (%) MoE One 14 1144 81 12 0.29 51 43 2 26 739 28 36 1.22 46 29 3 14 1397 100 10 0.28 50 36 4 14 478 34 29 1.13 47 26 * SwD Measured according to Procedure 1

The strain (MoD) of the crosslinked hyaluronate fibers obtained was measured by NMR spectroscopy as described above. Briefly, the signal intensity of the connected BDDE is related to the signal of the acetyl group of the HA. After the number of protons generating the signal is corrected, the molar ratio of the connected BDDE / HA can be calculated. Figures 2A (Form 1) and 2b (Formulation 2) show a 400 MHz ¹ H NMR spectrum of the enzymatically degraded HA-gel in deionized water (D ₂ O). The signal of the connected BDDE and the signal of the N-acetyl group are displayed in the spectrum. The MoD and CrR results for each formulation are shown in Table 6 together with the deformation efficiency (MoE).

The dried intermediate product of the crosslinked fibers was swollen to a defined HA-concentration as follows. Phosphate buffered saline was added to the dried HA-fibers to a concentration estimated to be about 20 mg HA / mL. Formulations 1 and 3 absorbed all of the injected solution, but Formulations 2 and 4 did not. The swollen gel was filled in a 1 mL glass syringe under sterile conditions. The filled syringes were sterilized using a wet heat treatment at 125 ° C and a final F _{0 of} about 20 minutes.

For the contents of the autoclaved syringe, HA concentration, gel content (GelC), swelling degree (SwD) and pH were determined according to the method described above. The results for the products after sterilization are shown in Table 7.

Formulation HA concentration (mg / mL) GelC (%) SwD * (g / g HA) pH One 18 96 95 7.5 2 32 ~ 99 25 7.6 3 19 95 87 7.6 4 36 ~ 99 23 7.5 * SwD Measured according to Procedure 3, corrected for gel content

In Tables 6 and 7, it is clearly stated that formulations 1 and 3 are less robust and less chemically deformable than Formulations 2 and 4. HA high concentrations of Formulations 2 and 4 are found to have low maximum water uptake and impossible to prepare gel products with low HA concentration for the gels produced under these conditions.

Formulations were analyzed for bioburden (injection plate method) and endotoxin (gel-coagulation method). All formulations were Bioburden 0 cfu / syringe and the endotoxin level was <0.21 EU / mL. All of the formulations met the requirements associated with biocompatibility purity experiments.

Microscopy

The swollen and autoclaved fibers were visualized by staining in a toluidine blue aqueous solution for 15 minutes. A micrograph is shown in Figures 3a-d. 3a: Formulation 1; 3b: Formulation 2; 3c: Formulation 3; And 3d: Formulation 4. Bar = 1 mm.

Stability check

The stability test of the formulation was carried out at 60 [deg.] C / ambient relative humidity (RH) for 14 days. All of the formulations showed good stability. Figure 4 shows the SwD increase over time at 60 占 (measured according to SwD procedure 3 and corrected for gel content). Formulation 1 (?); Formulation 2 (?); Formulation 3 (?); And Formulation 4 (x).

Biological evaluation

The biocompatibility of the formulation was investigated by subcutaneous injection in 24 Sprague-Dawley rats. Animals were anesthetized and then deprived of their back, and 1 mL of each formulation was subcutaneously injected. The injection was performed twice on both sides of the center line of the rats and the like. The animals were divided into two groups. One group was euthanized one week after injection and the other group was euthanized three weeks after injection. The animals were checked daily and recorded for signs of disease. The injected formulations were macroscopically and histologically acceptable in all skin samples. Signals of necrosis or acute inflammation were not observed. No tissue reaction symptoms or formation of granuloma were found other than a thin fibrous capsule (not shown) surrounding the implantation site of the formulation. The formulations could be concluded to be biocompatible in the rat.

Example 8 - Degradation of gels with various gel strengths by hyaluronidase

A hyaluronate solution was prepared as described in Example 1. After complete dissolution, the pH of the resulting hyaluronate solution was higher than 11.5 when measured as described above. The hyaluronate solution was pushed through a 0.5 mm diameter opening into a 99.55 ethanol liquid medium. The extruded hyaluronate solution immediately precipitated into hyaluronate fibers.

Approximately 1 g of the precipitated HA fiber was transferred to 60 mL of a crosslinked aqueous solution containing 70% ethanol, BDDE and NaOH. The hyaluronate was still in a precipitated state and retained its fibrous morphology. The crosslinking media obtained, including HA, had a pH greater than 11.5.

The HA fibers were crosslinked in an aqueous / organic crosslinking medium. Then, the obtained crosslinked hyaluronate fiber was taken from the crosslinking medium and neutralized by adding phosphoric acid thereto. The crosslinked HA fibers were dried in a vacuum chamber at room temperature under reduced pressure (~ 200 mbar) for about 2 hours.

The intermediate product thus precipitated of the crosslinked HA fibers was swollen to a defined HA concentration as follows. Phosphate buffered saline was added to the dried HA fibers to a concentration of about 20-40 mg HA / mL. A somewhat swollen gel was filled into a 10 mL plastic syringe. The filled syringes were sterilized by wet heat treatment at 123 ° C and the final F ₀ was approximately 20 minutes. The swelling degree (SwD procedure 2), the HA concentration, the HA minimum concentration (C _min ), the degree of deformation (MoD), the deformation efficiency (MoE) and the viscoelastic characteristics of the autoclaved syringe were determined according to the above-described method. All of the various products were subjected to hyaluronidase digestion according to "Enzymatic Degradability Determination" to verify that the biodegradability of HA was retained in the crosslinked product.

The results are shown in Tables 8A and 8B. The value of G '/ (G' + G ") is clearly> 70% in all formulations, so that they are all gels. In addition, the G 'value shows that the gel is hard, And the rheological properties, the gel is able to degrade by more than 99% by hyaluronidase over a wide range of gel strengths, indicating that the biodegradability of the natural HA is beneficially retained in the gel product according to the invention it means.

Table 8A

Crosslinking process Characteristic Sample Crosslinking concentration SwD * (mL / g) C _min (mg / mL) MoD (%) Decomposition by hyaluronidase (%) HA concentration
(mg / mL) One 1Ф 75 14 0.2 > 99 20 2 2Ф 26 39 0.4 > 99 17 3 ** 6Ф 17 59 1.9 > 99 44 4 8Ф 17 59 1.6 > 99 39

* Measured according to Procedure 2

** Bridging temperature 29 ℃

Table 8B

Sample G '1 Hz (Pa) G "1 Hz (Pa) G '0.1 Hz (Pa) G "0.1 Hz (Pa) G '1 Hz (%) * G '0.1 Hz (%) * One 202 90 117 47 69% 71% 2 435 45 674 36 91% 95% 3 7975 527 7207 689 94% 91% 4 6485 352 5914 478 95% 93%

 * Gel properties calculated with G '/ (G' + G ").

Example 9 - Comparative Example

Referring to Table 9A below, five hyaluronic acid (HA) gel samples were prepared according to Example 4 of EP 2 199 308 A1. Reference sample (FU0509: 1) according to the present invention was prepared using the crosslinking process described in Example 1 above.

                Table 9A - Crosslinking conditions

Sample HA Supplier Stirring during crosslinking Crosslinking temperature Reaction time
(h) One Shiseido Do not RT 16 h 2 Shiseido Magnet RT 16 h 3 Shiseido Do not 45 ° C 16 h 4 Shiseido Magnet 40 ℃ 16 h 5 Food Chemifa prop RT 16 h Reference sample Food Chemifa N / A RT 48 h

                  Table 9B - Results

Sample MoD (%) Gφ 0.1 Hz G "0.1 Hz Gφ 1 Hz G "1 Hz 0.34 57 50 174 110 2 0.53 58 37 141 80 3 1.05 78 24 127 48 4 0.79 32 13 58 28 5 1.70 145 6 157 10 Reference sample 0.36 853 40 932 53

Referring to Table 9B, a gel was obtained in all samples. The irregularly shaped gel particles provided by Comparative Samples 1-5 showed MoD values in the same range as the samples prepared by the method according to the present invention. However, the G ' and G "values were significantly lower in the Comparative Samples 1-5 compared to the samples prepared by the method according to the present invention. Because the present invention provides already harder gels at low MoD, Was determined to be more effective than the method described in EP 2 199 308 A1.

Comparative Samples 1-5 Each of the micrographs (50x magnification each of Figures 5a-e) shows that the size and shape distribution of the product is broad, meaning that the method disclosed in EP 2 199 308 A1 produces random sized particles do. On the other hand, a micrograph (FIG. 5f) of the product produced by the process according to the invention shows products of uniform size and shape.

Example 10 - Ethanol concentration in the precipitation step

The HA string precipitation effect was tested at various ethanol concentrations. Four ethanol aqueous solutions having concentrations of 65, 75, 85 and 95% w / w were prepared. HA solution (5% w / w) in ethanol (35% w / w) + 0.5% NaOH (w / w) was manually extruded onto a small plastic (PE) film, w) ethanol was immersed in each tank or immersed stepwise in 75% and 85% ethanol for 1 minute and then transferred to 99.5% ethanol.

The precipitated strings produced were collected and visually evaluated using a light microscope. Sensory feelings when handling strings were also evaluated. The higher the concentration of ethanol used in the precipitation bath, the smoother and flatter the surface, the stronger the curling, the fragile, and the more bundle structure, the string was observed.

Strings precipitated stepwise (75% -> 85% -> 99.5%) showed similar behavior to strings precipitated only at 75 w / w% or 85 w / w%. It was observed that HA was not completely precipitated when 65% ethanol was used under these conditions. The sample that was placed in 65% ethanol for 20 minutes and then completely precipitated in 99.5% ethanol had a completely different appearance from the other strings; It had no bundle structure and had a flat, thin band-like structure.

Example 11 - Arrangement of a desired shape on a hydrophobic surface

The effect of the precipitation method was tested by comparing the following two processes:

(I): 1) extrusion - 2) put on PE film - 3) precipitation in 99.5% ethanol (comparison, Example 10)

(II): 1) Extrusion - 2) Free fall with 99.5% ethanol - 3) Seal on PE film

The prepared precipitated strings were collected and visually evaluated with a light microscope. Sensory feelings when handling strings were also evaluated. In the first type of precipitation process (I), a smooth, uniform string was produced, but in the second type of precipitation process (II), a broken, uneven and stringy protruding string was produced.

Example 12 - Subcutaneous injection into rats

The in-vivo test was performed by subcutaneously injecting a gel formulation prepared in the same manner as described in Example 7 into a hairless rat (Sprague Dawley). As a result, the string form of the implanted gel (Fig. 6A) remained after 6 months in the subcutaneous region of the rat (Fig. 6B). Figure 6a shows a string (non-exterminated) stained with toluidine blue after dilution with Milli-RX and extrusion through an 18G cannula. Figure 6b shows the same string arrangement taken from the subcutaneous region of the rat after 6 months.

The shape of the string was judged to be retained not only after extrusion from the needle but also after 6 months in the rat.

Example 13 - Comparative Example

US 2012/0034462 A1 proposes without experimental evidence that a thin strand of an artificial HA gel can be prepared by passing through a solid mass of HA gel cross-linked through a sieve or mesh. To test this theory, a gel was prepared according to Example 1 (HA MW 3 MDa; BDDE 75 mg / g HA; temperature 50 ° C, 2 hours) of US 2012/0034462 A1. The hydrogel was passed once through a 32 [mu] m or 63 [mu] m mesh screen.

The structure of the resulting HA gel is shown in Figure 7 at 10x magnification: (A) 32 [mu] m mesh size; (B) 63 mu m mesh size (bar = 2 mm). The prepared particles did not show any orderly structure, and in particular were not of any elongated shape.

Claims (40)

A method for producing a molded crosslinked hyaluronic acid product,
(i) providing a hyaluronic acid substrate dissolved in a first liquid medium which is an aqueous solution, without crosslinking;
(ii) subjecting the hyaluronic acid substrate to a second liquid medium comprising, without crosslinking, at least one first water soluble organic solvent (s) in an amount that provides precipitation conditions for hyaluronic acid, thereby precipitating the hyaluronic acid substrate ; And
(iii) contacting the non-crosslinked precipitated hyaluronic acid substrate of the desired shape with one or more second organic solvent (s) having a pH of at least 11.5 and at least one multifunctional crosslinking agent (s) and an amount to provide precipitation conditions for hyaluronic acid Crosslinked hyaluronic acid product, in a third liquid medium containing a crosslinked hyaluronic acid,
Wherein step (i) and / or step (ii) further comprises arranging the hyaluronic acid substrate in a desired shape. The method according to claim 1,
Wherein the first two steps (i) and (ii) are carried out in the absence of a cross-linking agent,
Characterized in that the polyfunctional crosslinking agent (s) is added in the third crosslinking step (iii). 3. The method according to claim 1 or 2,
Wherein step (i) further comprises arranging the hyaluronic acid substrate solution in a desired shape on a hydrophobic surface;
Wherein precipitation of the shaped hyaluronic acid matrix in step (ii) occurs on the hydrophobic surface. The method of claim 3,
Characterized in that the hydrophobic surface is selected from fluorocarbons, polypropylene (PP), polyethylene terephthalate glycol-modified (PETG), polyethylene (PE) and polytetrafluoroethylene (PTFE). 5. The method according to any one of claims 1 to 4,
Characterized in that the shape is selected from the group consisting of particles, fibers, strings, strands, nets, films, discs and beads. 6. The method of claim 5,
Wherein said shape is a fiber,
Wherein the ratio of the length of the fibers to the width of the fibers is 10: 1 or more. 3. The method according to claim 1 or 2,
Wherein said step (ii) comprises extruding said hyaluronic acid substrate into a second liquid medium comprising said first water soluble organic solvent (s) in an amount which provides precipitation conditions for hyaluronic acid, whereby said extruded hyaluronic acid substrate To form precipitated fibers in the second liquid medium. 8. The method according to any one of claims 1 to 7,
Wherein the second liquid medium of step (ii) comprises between 0 vol% and 30 vol% of water and between 70 vol% and 100 vol% of the first water soluble organic solvent (s). 9. The method of claim 8,
Wherein the second liquid medium of step (ii) comprises 0 vol% to 10 vol% of water and 90 vol% to 100 vol% of the first water soluble organic solvent (s). 10. The method according to any one of claims 1 to 9,
Characterized in that the third liquid medium of step (iii) comprises from 0 vol% to 35 vol% of water, from 65 vol% to 100 vol% of the second organic solvent (s), and at least one multifunctional crosslinking agent (s) . 11. The method according to any one of claims 1 to 10,
Wherein the organic solvent (s) are individually selected from one or more lower alkyl alcohol (s). 13. The method according to any one of claims 1 to 12,
Wherein the aqueous solution of step (i) comprises from 40% to 100% by volume of water and from 0% to 60% by volume of lower alkyl alcohol (s). 13. The method according to claim 11 or 12,
Wherein the lower alkyl alcohol is ethanol. 14. The method according to any one of claims 1 to 13,
Wherein the multifunctional crosslinking agent (s) is selected from the group consisting of divinyl sulfone, multiepoxide and diepoxide. 15. The method of claim 14,
Wherein the polyfunctional crosslinking agent (s) is individually selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxy octane &Lt; / RTI &gt; 16. The method of claim 15,
Wherein the polyfunctional crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). 17. The method according to any one of claims 1 to 16,
(iv) treating the precipitated, crosslinked hyaluronic acid product in non-precipitation conditions; And
(v) separating the crosslinked hyaluronic acid product into a non-precipitated form. 18. The method of claim 17,
Wherein said step (v) further comprises sterilizing said crosslinked hyaluronic acid product. Wherein the degree of modification is from 1 to 40 units of crosslinking agent per 1000 units of disaccharide and from 4 mL to 300 mL of swelling degree per gram of hyaluronic acid and having a modification efficiency of 10 or more, Hyaluronic acid product. 20. The method of claim 19,
Characterized in that the product has a shape selected from the group consisting of particles, fibers, strings, strands, nets, films, discs and beads. 21. The method of claim 20,
Wherein the product is a fiber,
Characterized in that the ratio of the length of the fibers to the width of the fibers is at least 10: 1. 22. The method according to claim 20 or 21,
Wherein the product is a fiber,
Characterized in that the longitudinal extension of the fibers is greater than 2 mm. 23. The method according to any one of claims 19 to 22,
Wherein the degree of swelling is from 15 mL to 180 mL per g of hyaluronic acid. 24. The method according to any one of claims 19 to 23,
Wherein the strain efficiency is in the range of 20 to 190. &lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; 25. The method according to any one of claims 19 to 24,
The shaped crosslinked hyaluronic acid product is characterized in that the crosslinking ratio is 35% or more. 26. The method according to any one of claims 19 to 25,
Wherein the crosslinked hyaluronic acid product is crosslinked with at least one multifunctional crosslinking agent (s), individually selected from the group consisting of polyvinylpyrrolidone, divinyl sulfone, multiepoxide and diepoxide. 27. The method of claim 26,
Wherein the polyfunctional crosslinking agent (s) is individually selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethanediol diglycidyl ether (EDDE) and diepoxy octane Lt; RTI ID = 0.0 &gt; hyaluronic &lt; / RTI &gt; acid product. 28. The method of claim 27,
Wherein the polyfunctional crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE). 29. The method according to any one of claims 19 to 28,
Wherein the product is sterilized. &Lt; RTI ID = 0.0 &gt; 18. &lt; / RTI &gt; 18. A shaped crosslinked hyaluronic acid product produced by the process according to any one of claims 1 to 18. 30. An aqueous composition comprising a shaped crosslinked hyaluronic acid product according to any one of claims 19 to 30 and optionally a buffering agent. Use of a shaped crosslinked hyaluronic acid product according to any one of claims 19 to 30 or an aqueous composition according to claim 31 for cosmetic surgery. 33. The method of claim 32,
&Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt; human contour. 30. A shaped crosslinked hyaluronic acid product according to any one of claims 19 to 30 or an aqueous composition according to claim 31 for use as a medicament or a medical device. 30. A shaped crosslinked hyaluronic acid product according to any one of claims 19 to 30 or an aqueous composition according to claim 31 for use in cosmetic or medical surgery. 36. The method of claim 35,
&Lt; / RTI &gt; wherein the shaped crosslinked hyaluronic acid product or aqueous composition is for use in a cosmetic surgery selected from skin rash and human contour. 36. The method of claim 35,
Wherein the shaped crosslinked hyaluronic acid product or aqueous composition is for use in medical surgery selected from skin repercussions, human contouring, prevention of tissue adhesion, channel formation, incontinence treatment and orthopedic application. 30. A shaped crosslinked hyaluronic acid product according to any one of claims 19 to 30 or an aqueous composition according to claim 31 for use in drug delivery. 30. A pre-filled syringe pre-filled with a sterilized, shaped crosslinked hyaluronic acid product according to any of claims 19 to 30 or a sterile aqueous composition according to claim 31. Comprising the step of administering a shaped crosslinked hyaluronic acid product according to any one of claims 19 to 30 or an aqueous composition according to claim 31 to a subject in need thereof for a cosmetic or medical operation A method of treating an individual.

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